Patent application title: CHIMERIC INHIBITORY RECEPTOR
Inventors:
Russell Morrison Gordley (San Francisco, CA, US)
Nicholas Frankel (South San Francisco, CA, US)
IPC8 Class: AC07K1628FI
USPC Class:
1 1
Class name:
Publication date: 2022-09-15
Patent application number: 20220289842
Abstract:
Provided herein are chimeric inhibitory receptor constructs and
compositions, and cells comprising the same. Also provided are methods of
using chimeric inhibitory receptor constructs and compositions, and cells
comprising the same.Claims:
1. A chimeric inhibitory receptor comprising: an extracellular ligand
binding domain; a membrane localization domain, wherein the membrane
localization domain comprises a transmembrane domain; and an enzymatic
inhibitory domain, wherein the enzymatic inhibitory domain inhibits
immune receptor activation when proximal to an immune receptor.
2. The chimeric inhibitory receptor of claim 1, wherein the enzymatic inhibitory domain comprises an enzyme catalytic domain, and wherein the enzyme catalytic domain is from an enzyme selected from the group consisting of: CSK, SHP-1, SHP-2, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, ZAP70, and RasGAP.
3. The chimeric inhibitory receptor of claim 1 or claim 2, wherein the enzymatic inhibitory domain comprises at least a portion of an extracellular domain, a transmembrane domain, and/or an intracellular domain.
4. The chimeric inhibitory receptor of any one of claims 1-3, wherein the extracellular ligand binding domain comprises an antibody, or antigen-binding fragment thereof, optionally wherein the or antigen-binding fragment is a a F(ab) fragment, a F(ab') fragment, or a single chain variable fragment (scFv).
5. The chimeric inhibitory receptor of any one of claims 1-4, wherein the extracellular ligand binding domain binds to a ligand that is not expressed on a tumor cell and/or the ligand is expressed on cells of a healthy tissue.
6. The chimeric inhibitory receptor of any one of claims 1-5, wherein the extracellular ligand binding domain comprises a dimerization domain, optionally wherein the ligand further comprises a cognate dimerization domain.
7. The chimeric inhibitory receptor of any one of claims 1-6, wherein the membrane localization domain further comprises at least a portion of an extracellular domain and/or at least a portion of an intracellular domain, and optionally wherein the transmembrane domain is selected from the group consisting of: a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain from a LAT mutant, a BTLA transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIR1 transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAG3 transmembrane domain, a LAIR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, an SIRPa transmembrane domain, an HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain.
8. The chimeric inhibitory receptor of any one of claims 1-7, wherein the chimeric inhibitory receptor further comprises one or more intracellular inhibitory co-signaling domains, optionally wherein the one or more intracellular inhibitory co-signaling domains comprise one or more ITIM-containing proteins, or fragments thereof, selected from the group consisting of: PD-1, CTLA4, TIGIT, BTLA, and LAIR1; and/or the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or fragments thereof, selected from the group consisting of: GRB-2, Dok-1, Dok-2, SLAP1, SLAP2, LAG3, HAVR, GITR, and PD-L1.
9. The chimeric inhibitory receptor of any one of claims 1-8, wherein the extracellular ligand binding domain is linked to the membrane localization domain through an extracellular linker region, optionally wherein the extracellular linker region is positioned between the extracellular ligand binding domain and membrane localization domain and operably and/or physically linked to each of the extracellular ligand binding domain and the membrane localization domain, optionally wherein the extracellular linker region is derived from a protein selected from the group consisting of: CD8alpha, CD4, CD7, CD28, IgG1, IgG4, FcgammaRIIIalpha, LNGFR, and PDGFR or comprises an amino acid sequence selected from the group consisting of: TABLE-US-00012 (SEQ ID NO: 29) GGS, (SEQ ID NO: 30) GGSGGS, (SEQ ID NO: 31) GGSGGSGGS, (SEQ ID NO: 32) GGSGGSGGSGGS, (SEQ ID NO: 33) GGSGGSGGSGGSGGS, (SEQ ID NO: 34) GGGS, (SEQ ID NO: 35) GGGSGGGS, (SEQ ID NO: 36) GGGSGGGSGGGS, (SEQ ID NO: 37) GGGSGGGSGGGSGGGS, (SEQ ID NO: 38) GGGSGGGSGGGSGGGSGGGS, (SEQ ID NO: 39) GGGGS, (SEQ ID NO: 40) GGGGSGGGGS, (SEQ ID NO: 41) GGGGSGGGGSGGGGS, (SEQ ID NO: 42) GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 43) GGGGSGGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 46) AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP, (SEQ ID NO: 47) ESKYGPPCPSCP, (SEQ ID NO: 48) ESKYGPPAPSAP, (SEQ ID NO: 49) ESKYGPPCPPCP, (SEQ ID NO: 50) EPKSCDKTHTCP, (SEQ ID NO: 51) AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN, (SEQ ID NO: 52) TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD, (SEQ ID NO: 53) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSAT EPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEA GSGLVFSCQDKQNTVCEECPDGTYSDEADAEC, (SEQ ID NO: 54) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC, and (SEQ ID NO: 55) AVGQDTQEVIVVPHSLPFKV.
10. The chimeric inhibitory receptor of any one of claims 1-9, wherein the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the membrane localization domain and the enzymatic inhibitory domain and operably and/or physically linked to each of the membrane localization domain and the enzymatic inhibitory domain, optionally, wherein the intracellular spacer region comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:46), ESKYGPPCPSCP (SEQ ID NO:47), ESKYGPPAPSAP (SEQ ID NO:48), ESKYGPPCPPCP (SEQ ID NO:49), EPKSCDKTHTCP (SEQ ID NO:50), AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:51), TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:52), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEP CKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSG LVFSCQDKQNTVCEECPDGTYSDEADAEC (SEQ ID NO:53), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC (SEQ ID NO:54), and AVGQDTQEVIVVPHSLPFKV (SEQ ID NO:55).
11. The chimeric inhibitory receptor of any one of claims 1-10, wherein the immune receptor is a chimeric immune receptor, optionally wherein the immune receptor is a chimeric antigen receptor (CAR), a naturally-occurring antigen receptor, optionally wherein the immune receptor is selected from the group consisting of a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
12. A nucleic acid encoding the chimeric inhibitory receptor of any one of claims 1-11.
13. A vector comprising the nucleic acid of claim 12.
14. A genetically engineered cell comprising the nucleic acid of claim 12, the vector of claim 13 or expressing the chimeric inhibitory receptor of any one of claims 1-11.
15. A genetically engineered cell expressing a chimeric inhibitory receptor, wherein the chimeric inhibitory receptor comprises: an extracellular ligand binding domain; a membrane localization domain, wherein the membrane localization domain comprises a transmembrane domain; and an enzymatic inhibitory domain, wherein the inhibitory domain inhibits immune receptor activation when proximal to an immune receptor, optionally wherein the cell further comprises an immune receptor, optionally wherein the immune receptor is a chimeric antigen receptor or a naturally-occurring antigen receptor, optionally wherein the immune receptor is selected from the group consisting of: a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor, optionally wherein the chimeric inhibitory receptor inhibits immune receptor activation upon ligand binding.
16. The engineered cell of claim 14 or claim 15, wherein the cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.
17. A pharmaceutical composition comprising the engineered cell of any one of claims 14-16 and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or combination thereof.
18. A method of inhibiting immune receptor activation, comprising: contacting the engineered cell of any one of claims 14-16 or the pharmaceutical composition of claim 17 with a cognate ligand under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein, when localized proximal to an immune receptor expressed on a cell membrane of the engineered cell, the chimeric inhibitory inhibits immune receptor activation.
19. A method of preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, comprising: administering the engineered cell of any one of claims 14-16 or the pharmaceutical composition of claim 17 to a subject in need of such treatment.
20. A method of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, comprising: contacting the engineered cell of any one claims 14-16 or the pharmaceutical composition of claim 17 or the pharmaceutical composition of claim 17 with a cognate ligand of the chimeric inhibitory receptor under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein upon binding of the ligand to the chimeric inhibitory receptor, the enzymatic inhibitory domain prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/889,324, filed Aug. 20, 2019, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Month XX, 20XX, is named XXXXXUS_sequencelisting.txt, and is X,XXX,XXX bytes in size.
BACKGROUND
[0003] Chimeric antigen receptors (CARs) enable targeted in vivo activation of immunomodulatory cells, such as T cells. These recombinant membrane receptors have an antigen-binding domain and one or more signaling domains (e.g., T cell activation domains). These special receptors allow the T cells to recognize a specific protein antigen on tumor cells and induce T cell activation and signaling pathways. Recent results of clinical trials with chimeric receptor-expressing T cells have provided compelling support of their utility as agents for cancer immunotherapy. However, despite these promising results, a number of side effects associated the CAR T-cell therapeutics were identified, raising significant safety concerns. One side effect is "on-target but off-tissue" adverse events from TCR and CAR engineered T cells, in which a CAR T cell binds to its ligand outside of the target tumor tissue and induces an immune response. Therefore, the ability to identify appropriate CAR targets is important for effectively targeting and treating the tumor without damaging normal cells that express the same target antigen. The ability to regulate an appropriate response to targets and reduce off-target side effects is important in other immune receptor systems as well, such as TCRs, engineered TCRs, and chimeric TCRs.
[0004] Inhibitory chimeric antigen receptors (also known as iCARs) are protein constructions that inhibit or reduce immunomodulatory cell activity after binding their cognate ligands on a target cell. Current iCAR designs leverage PD-1 intracellular domains for inhibition, but have proven difficult to reproduce. Thus, alternative inhibitory domains for use in iCARs are needed. Appropriate inhibitory domains, strategies, and constructs for immune receptor systems are also needed.
SUMMARY
[0005] Provided herein, in some aspects, are chimeric inhibitory receptors that include: an extracellular ligand binding domain; a membrane localization domain that includes a transmembrane domain; and an enzymatic inhibitory domain that inhibits immune receptor activation when proximal to an immune receptor.
[0006] Provided herein, in other aspects, are nucleic acids encoding at least one chimeric inhibitory receptor of the present disclosure. In some embodiments, the nucleic acid encoding the at least one chimeric inhibitory receptor is a vector.
[0007] In other aspects, genetically engineered cells are provided including a nucleic acid, such as a vector, encoding at least one chimeric receptor of the present disclosure or that express a chimeric inhibitory receptor of the present disclosure. In some aspects, genetically engineered cells expressing a chimeric inhibitory receptor are provided, wherein the chimeric inhibitory receptor includes: an extracellular ligand binding domain; a membrane localization domain, wherein the membrane localization domain comprises a transmembrane domain; and an enzymatic inhibitory domain, wherein the inhibitory domain inhibits immune receptor activation when proximal to an immune receptor.
[0008] In still other aspects, methods are provided for inhibiting immune receptor activation in genetically engineered cells of the present disclosure.
[0009] In yet other aspects, methods are provided for utilizing genetically engineered cells or pharmaceutical compositions of the present disclosure to reduce an immune response and/or treat an autoimmune disease.
[0010] In other aspects, pharmaceutical composition including the engineered cell of any one of the compositions provided for herein and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or combination thereof.
[0011] In some embodiments, the extracellular ligand binding domain binds to a ligand selected from: a protein complex, a protein, a peptide, a receptor-binding domain, a nucleic acid, a small molecule, and a chemical agent.
[0012] In some embodiments, the extracellular ligand binding domain includes an antibody, or antigen-binding fragment thereof. In some embodiments, the extracellular ligand binding domain includes a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).
[0013] In some of these embodiments, the ligand is a tumor-associated antigen. In some of these embodiments, the ligand is not expressed on a tumor cell. In some of these embodiments, the ligand is expressed on a non-tumor cell. In some of these embodiments, the ligand is expressed on cells of a healthy tissue.
[0014] In some embodiments, the extracellular ligand binding domain includes a dimerization domain. In some embodiments, the ligand further includes a cognate dimerization domain.
[0015] In some embodiments, the ligand is a cell surface ligand. In some embodiments, the cell surface ligand is expressed on a cell that further expresses a cognate ligand of the immune receptor.
[0016] In some embodiments, the membrane localization domain of a chimeric receptor of the present disclosure further includes at least a portion of an extracellular domain. In some embodiments, the membrane localization domain further includes at least a portion of an intracellular domain. In some embodiments, the membrane localization domain further includes at least a portion of an extracellular domain and at least a portion of an intracellular domain.
[0017] In some embodiments, the membrane localization domain includes a transmembrane domain selected from the group consisting of: a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain from a LAT mutant, a BTLA transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIR1 transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAGS transmembrane domain, a LAIR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, an SIRPa transmembrane domain, an HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain. In some embodiments, the membrane localization domain further includes at least a portion of a corresponding extracellular domain and/or at least a portion of a corresponding intracellular domain. In some embodiments, the LAT mutant is a LAT(CA) mutant.
[0018] In some embodiments, the membrane localization domain directs and/or segregates the chimeric inhibitory receptor to a domain of a cell membrane. In some embodiments, the membrane localization domain localizes the chimeric inhibitory receptor to a lipid raft or a heavy lipid raft. In some embodiments, the membrane localization domain interacts with one or more cell membrane components localized in a domain of a cell membrane. In some embodiments, the membrane localization domain is sufficient to mitigate constitutive inhibition of immune receptor activation by the enzymatic inhibitory domain in the absence of the extracellular ligand binding domain binding a cognate ligand.
[0019] In some embodiments, the membrane localization domain mediates localization of the chimeric inhibitory receptor to a domain of a cell membrane that is distinct from domains of the cell membrane occupied by one or more components of an immune receptor in the absence of the extracellular ligand binding domain binding a cognate ligand.
[0020] In some embodiments, the membrane localization domain further includes proximal protein fragments. In some embodiments, the membrane localization domain further includes one or more intracellular inhibitory co-signaling domains. In some embodiments, the one or more intracellular inhibitory co-signaling domains of a chimeric protein include one or more ITIM-containing proteins, or fragments thereof. In some embodiments, the one or more ITIM-containing proteins, or fragments thereof, are selected from PD-1, CTLA4, TIGIT, BTLA, and LAIR1. In some embodiments, the one or more intracellular inhibitory co-signaling domains include one or more non-ITIM scaffold proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP1, SLAP2, LAGS, HAVR, GITR, and PD-L1.
[0021] In some embodiments, the extracellular ligand binding domain of a chimeric inhibitory receptor of the present disclosure is linked to the membrane localization domain through an extracellular linker region.
[0022] In some embodiments, the extracellular linker region is positioned between the extracellular ligand binding domain and membrane localization domain and operably and/or physically linked to each of the extracellular ligand binding domain and the membrane localization domain. In some embodiments, the extracellular linker region is derived from a protein selected from the group consisting of: CD8alpha, CD4, CD7, CD28, IgG1, IgG4, FcgammaRIIIalpha, LNGFR, and PDGFR. In some embodiments, the extracellular linker region comprises an amino acid sequence selected from the group consisting of: AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:46), ESKYGPPCPSCP (SEQ ID NO:47), ESKYGPPAPSAP (SEQ ID NO:48), ESKYGPPCPPCP (SEQ ID NO:49), EPKSCDKTHTCP (SEQ ID NO:50), AAAFVPVFLPAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:51), TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:52), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCT ECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ NTVCEECPDGTYSDEADAEC (SEQ ID NO:53), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC (SEQ ID NO:54), and AVGQDTQEVIVVPHSLPFKV (SEQ ID NO:55). In some embodiments, the extracellular linker region comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45).
[0023] In some embodiments, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the membrane localization domain and the enzymatic inhibitory domain and operably and/or physically linked to each of the membrane localization domain and the enzymatic inhibitory domain. In some embodiments, the intracellular spacer region comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45). In some embodiments, the intracellular spacer region comprises an amino acid sequence selected from the group consisting of:
TABLE-US-00001 (SEQ ID NO: 46) AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP, (SEQ ID NO: 47) ESKYGPPCPSCP, (SEQ ID NO: 48) ESKYGPPAPSAP, (SEQ ID NO: 49) ESKYGPPCPPCP, (SEQ ID NO: 50) EPKSCDKTHTCP, (SEQ ID NO: 51) AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN, (SEQ ID NO: 52) TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD. (SEQ ID NO: 53) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSAT EPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEA GSGLVFSCQDKQNTVCEECPDGTYSDEADAEC, (SEQ ID NO: 54) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC, and (SEQ ID NO: 55) AVGQDTQEVIVVPHSLPFKV.
[0024] In some embodiments, the enzymatic inhibitory domain of a chimeric inhibitory receptor of the present disclosure includes at least a portion of an extracellular domain, a transmembrane domain, and/or an intracellular domain. In some embodiments, the enzymatic inhibitory domain includes an enzyme catalytic domain.
[0025] In some embodiments, the enzymatic inhibitory domain includes at least a portion of an enzyme. In some embodiments, the portion of the enzyme includes an enzyme domain or an enzyme fragment. In some embodiments, the portion of the enzyme is a catalytic domain of the enzyme.
[0026] In some embodiments, the enzyme is selected from the group consisting of: CSK, SHP-1, SHP-2, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, ZAP70, and RasGAP.
[0027] In some embodiments, the enzymatic inhibitory domain is derived from CSK. In some embodiments, the enzymatic inhibitory domain comprises a CSK protein with a SRC homology 3 (SH3) deletion.
[0028] In some embodiments, the enzymatic inhibitory domain is derived from SHP-1. In some embodiments, the enzymatic inhibitory domain comprises a protein tyrosine phosphatase (PTP) domain.
[0029] In some embodiments, the enzymatic inhibitory domain is derived from SHP-2.
[0030] In some embodiments, the enzymatic inhibitory domain is derived from PTEN.
[0031] In some embodiments, the enzymatic inhibitory domain is derived from CD45.
[0032] In some embodiments, the enzymatic inhibitory domain is derived from CD148.
[0033] In some embodiments, the enzymatic inhibitory domain is derived from PTP-MEG1.
[0034] In some embodiments, the enzymatic inhibitory domain is derived from PTP-PEST.
[0035] In some embodiments, the enzymatic inhibitory domain is derived from c-CBL.
[0036] In some embodiments, the enzymatic inhibitory domain is derived from CBL-b.
[0037] In some embodiments, the enzymatic inhibitory domain is derived from PTPN22.
[0038] In some embodiments, the enzymatic inhibitory domain is derived from LAR.
[0039] In some embodiments, the enzymatic inhibitory domain is derived from PTPH1.
[0040] In some embodiments, the enzymatic inhibitory domain is derived from SHIP-1. In some embodiments, the enzymatic inhibitory domain comprises a protein tyrosine phosphatase (PTP) domain.
[0041] In some embodiments, the enzymatic inhibitory domain is derived from ZAP70. In some embodiments, the enzymatic inhibitory domain comprises a SRC homology 1 (SH1) domain, a SRC homology 2 (SH2) domain, or an SH1 domain and an SH2 domain. In some embodiments, the enzymatic inhibitory domain comprises a ZAP70 protein with a kinase domain deletion. In some embodiments, wherein the enzymatic inhibitory domain comprises a mutant ZAP70 protein with a Tyr492Phe amino acid substitution, a Tyr493Phe amino acid substitution, or a Tyr492Phe amino acid substitution and a Tyr493Phe amino acid substitution.
[0042] In some embodiments, the enzymatic inhibitory domain is derived from RasGAP.
[0043] In some embodiments, the enzymatic inhibitory domain includes one or more modifications that modulate basal inhibition. In some embodiments, the one or more modifications reduce basal inhibition. In other embodiments, the one or more modifications increase basal inhibition.
[0044] In some embodiments, the enzymatic inhibitory domain inhibits immune receptor activation upon recruitment of the chimeric inhibitory receptor proximal to an immune receptor.
[0045] In some embodiments, the immune receptor is a chimeric immune receptor. In some embodiments, the immune receptor is a chimeric antigen receptor. In some embodiments, the immune receptor is a naturally-occurring immune receptor. In some embodiments, the immune receptor is a naturally-occurring antigen receptor.
[0046] In some embodiments, the immune receptor is selected from a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
[0047] In some embodiments, the immune receptor is a T cell receptor.
[0048] In some embodiments, a genetically engineered cell of the present disclosure further includes at least one immune receptor. In some embodiments, the at least one immune receptor is a chimeric immune receptor. In some embodiments, the at least one immune receptor is a chimeric antigen receptor. In some embodiments, the at least one immune receptor is a naturally-occurring immune receptor. In some embodiments, the at least one immune receptor is a naturally-occurring antigen receptor. In some embodiments, the at least one immune receptor is selected from a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
[0049] In some embodiments, a chimeric inhibitory receptor of the present disclosure inhibits immune receptor activation upon ligand binding when proximal to the immune receptor.
[0050] In some embodiments, the ligand is a cell surface ligand. In some embodiments, the cell surface ligand is expressed on a cell that further expresses a cognate immune receptor ligand. In some embodiments, ligand binding to the chimeric inhibitory receptor and cognate immune receptor ligand binding to the immune receptor localizes the chimeric inhibitory receptor proximal to the immune receptor. In some embodiments, localization of the chimeric inhibitory receptor proximal to the immune receptor inhibits immune receptor activation.
[0051] In some embodiments, the cell is a T cell. In some embodiments, the immune receptor is a T cell receptor. In some embodiments, immune receptor activation is T cell activation.
[0052] In some embodiments, a genetically engineered cell of the present disclosure is an immunomodulatory cell. In some embodiments, the immunomodulatory cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.
[0053] In some embodiments, the cell is autologous. In some embodiments, the cell is allogeneic.
[0054] Also provided herein are methods of inhibiting immune receptor activation. The methods include: contacting a genetically engineered cell or a pharmaceutical composition disclosed herein under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein, when localized proximal to an immune receptor expressed on a cell membrane of the engineered cell, the chimeric inhibitory inhibits immune receptor activation.
[0055] Also provided herein are methods for reducing an immune response. The methods include: administering a genetically engineered cell or a pharmaceutical composition disclosed herein to a subject in need of such treatment.
[0056] Also provided herein are methods for preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell. The methods include: administering a genetically engineered cell or a pharmaceutical composition disclosed herein to a subject in need of such treatment.
[0057] Also provided herein are methods for preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell. The methods include: contacting a genetically engineered cell or a pharmaceutical composition disclosed herein with a cognate ligand of the chimeric inhibitory receptor under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein, upon binding of the ligand to the chimeric inhibitory receptor, the enzymatic inhibitory domain prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.
[0058] Also provided herein are methods of treating an autoimmune disease or disease treatable by reducing an immune response. The methods include: administering a genetically engineered cell or a pharmaceutical composition disclosed herein to a subject in need of such treatment.
[0059] These and other aspects are descried in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.
[0061] FIG. 1. Schematic depicting a mechanism whereby a chimeric inhibitory receptor of the present disclosure blocks T cell activation.
[0062] FIG. 2. Schematic depicting a composition of certain embodiments of a chimeric inhibitory receptor. ELBD: Extracellular Ligand Binding Domain--examples include, but are not limited to, scFv (e.g., against tumor antigen), natural receptor/ligand domains, and orthogonal dimerization domains (e.g., leucine zippers that could engage with a soluble targeting molecule); MLD: Membrane Localization Domain (optionally including proximal intra- and extra-cellular segments involved in localization to sub-domains of the cell membrane (e.g., lipid rafts)--examples include, but are not limited to, the transmembrane domains of LAX, CD25, CD7 (Pavel Otahal et al., Biochim Biophys Acta. 2011 February; 1813(2):367-76) and mutants of LAT (e.g. LAT(CA); see e.g., Kosugi A., et al. Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 June; 14(6): 669-80, the entirety of which is incorporated herein); EID: Enzymatic Inhibitory Domain (e.g., enzymes that inhibit the native T cell activation cascade, including domains, fragments, or mutants of enzymes, selected to maximize efficacy and minimize basal inhibition)--examples include, but are not limited to, CSK (Pavel Otahal et al., Biochim Biophys Acta. 2011 February; 1813(2):367-76), SHP-1 (see e.g., Kosugi A., et al. Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 June; 14(6): 669-80), PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, LYP/Pep/PTPN22, LAR, PTPH1, SHIP-1, RasGAP (see e.g., Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 September; 137(1): 1-19, the entirety of which is incorporated herein).
[0063] FIG. 3. Schematic depicting a composition of certain embodiments of a chimeric inhibitory receptor (e.g., an "extended" chimeric inhibitory receptor). ELBD, MLD, and EID as described for FIG. 2. SID: Scaffold Inhibitory Domain--examples include, but are not limited to, ITIM containing protein domains (e.g. cytoplasmic tails of PD-1, CTLA4, TIGIT, BTLA, and/or LAIR1), or fragment(s) thereof) and non-ITIM scaffold protein domains, or fragment(s) thereof, that inhibit T cell activation, including GRB-2, Dok-1, Dok-2, SLAP, LAGS, HAVR, GITR, and PD-L1.
[0064] FIG. 4. Schematic illustrating a NOT-gate aCAR/iCAR system. A T cell was engineered to express an anti-CD19 iCAR, including a CSK domain as the EID domain, to inhibit signaling of a co-expressed aCAR that included a CD28-CD3 intracellular signaling domain. Target k562 cells were engineered to express a cognate antigen for an aCAR (CD20) or engineered to express both the cognate antigen for the aCAR (CD20) and a cognate antigen for an iCAR (CD19).
[0065] FIG. 5. Representative flow-cytometry plots demonstrating expression of iCAR construct anti-CD19_scFv-Csk fusions at levels detectable above unmodified cells following transduction of CD4+ and CD8+ T cells without subsequent enrichment.
[0066] FIG. 6. Expression profiles as assessed by flow-cytometry for aCAR and iCAR constructs. Shown is: aCAR+=cells that express the aCAR (w/ and w/out iCAR) [first column]; iCAR+=cells that express the iCAR (w/ and w/out the aCAR) [second column]; and dual+=cells that express both the aCAR and iCAR [third column].
[0067] FIG. 7. Efficacy of iCAR inhibition of aCAR signaling as assessed by killing efficiency, represented as ratio of killing CD19/CD20 targets cells to CD20-only target cells. Shown is: transduction with an aCAR construct only (left column); co-transduction of T cells with an iCAR possessing a CSK enzymatic inhibitory domain (iCAR31) and an aCAR (middle column); and co-transduction of T cells with an iCAR possessing a CSK enzymatic inhibitory domain including an SH3 deletion (iCAR26) and an aCAR (right column).
DETAILED DESCRIPTION
Definitions
[0068] Terms used in the claims and specification are defined as set forth below unless otherwise specified.
[0069] The term "chimeric inhibitory receptor" or "inhibitory chimeric antigen receptor" or "inhibitory chimeric receptor" as used herein refers to a polypeptide or a set of polypeptides, which when expressed in a cell, such as an immune effector cell, provides the cell with specificity for a target cell and the ability to negatively regulate intracellular signal transduction. An chimeric inhibitory receptor may also be called an "iCAR."
[0070] The term "tumor-targeting chimeric receptor" or "activating chimeric receptor" refers to activating chimeric receptors, tumor-targeting chimeric antigen receptors (CARs), or engineered T cell receptors having architectures capable of inducing signal transduction or changes in protein expression in the activating chimeric receptor-expressing cell that results in the initiation of an immune response. A tumor targeting chimeric receptor may also be called an "aCAR."
[0071] The term, "transmembrane domain" as used herein, refers to a domain that spans a cellular membrane. In some embodiments, a transmembrane domain comprises a hydrophobic alpha helix.
[0072] The term "tumor" refers to tumor cells and the associated tumor microenvironment (TME). In some embodiments, tumor refers to a tumor cell or tumor mass. In some embodiments, tumor refers to the tumor microenvironment.
[0073] The term "not expressed" refers to expression that is at least 2-fold lower than the level of expression in non-tumor cells that would result in activation of the tumor-targeting chimeric antigen receptor. In some embodiments, the expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more lower than the level of expression in non-tumor cells that would result in activation of the tumor-targeting chimeric antigen receptor.
[0074] The term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
[0075] The term "in situ" refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.
[0076] The term "in vivo" refers to processes that occur in a living organism.
[0077] The term "mammal" as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
[0078] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
[0079] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0080] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
[0081] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
[0082] The term "sufficient amount" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.
[0083] The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.
[0084] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.
[0085] Chimeric Inhibitory Receptors
[0086] Provided herein are chimeric inhibitory receptors that are useful, inter alia, as a NOT logic gate for controlling immune cell activity. The chimeric inhibitory receptors include an extracellular ligand binding domain, a membrane localization domain including a transmembrane domain; and an enzymatic inhibitory domain. In some embodiments, the enzymatic inhibitory domain inhibits immune receptor activation upon recruitment of a chimeric inhibitory receptor of the present disclosure to be proximal to an immune receptor. Without wishing to be bound by theory, binding between the chimeric inhibitory receptor and its cognate ligand generally mediates spatial recruitment of the enzymatic inhibitory domain to be proximal to the immune receptor and/or downstream signaling complexes such that the enzymatic inhibitory domain is capable of negatively regulating an intracellular signal transduction cascade. Proximal can include two molecules (e.g., proteins or protein domains) physically interacting. Proximal can include two molecules being sufficiently physically close to operably interact with one another. Proximal can include two molecules physically or operably interacting with a shared intermediary molecule, e.g., a scaffold protein. Proximal can include two molecules physically or operably interacting with a shared complex, e.g., a signaling cascade. Proximal can include two molecules physically interacting for a duration of time to operably interact with one another. Proximal can include two molecules being sufficiently physically close for a duration of time to operably interact with one another. Proximal can include two molecules physically or operably interacting for a duration of time with a shared intermediary molecule, e.g., a scaffold protein. Proximal can include two molecules physically or operably interacting for a duration of time with a shared complex, e.g., a signaling cascade. Durations of time mediating operable interactions generally refers to interactions longer than stochastic interactions and can include sustained physical proximity, for example sustained ligand-mediated localization to a distinct domain of a cell membrane (e.g., an immunological synapse). Proximal to an immune receptor can include localization to a cellular environment allowing direct inhibition of the signaling activity of the immune receptor. Proximal to an immune receptor can include localization to a cellular environment allowing inhibition of an intracellular signal transduction cascade mediated by the immune receptor. The disclosed chimeric inhibitory receptors thus can be engineered to contain appropriate extracellular ligand binding domains that will reduce intracellular signaling, such as immune responses, in the presence of the cognate ligand. In some embodiments, the ligand is located on a cell surface. In some embodiments, the ligand is an agent that is not on a cell surface, such as a small molecule, secreted factor, environmental signal or other soluble and/or secreted agent that mediates spatial recruitment of the enzymatic inhibitory domain to be proximal to the immune receptor, such as a cross-linking reagent, a small molecule that mediates heterodimerization of protein domains, or antibody, each that can mediate spatial recruitment of the enzymatic inhibitory domain to be proximal to the immune receptor. Uses of the chimeric inhibitory receptors include, but are not limited to, reducing immune responses, controlling T cell activation, controlling CAR-T responses, and treating autoimmune diseases or any disease that is treatable by reducing immune responses.
[0087] Provided herein, in some aspects, are chimeric inhibitory receptors comprising: an extracellular ligand binding domain; a membrane localization domain, wherein the membrane localization domain comprises a transmembrane domain; and an enzymatic inhibitory domain, wherein the enzymatic inhibitory domain inhibits immune receptor activation when proximal to an immune receptor.
Enzymatic Inhibitory Domains
[0088] As used herein, the term "enzymatic inhibitory domain" refers to a protein domain having an enzymatic function that inhibits an intracellular signal transduction cascade, for example a native T cell activation cascade. For example, enzymatic inhibitory domains can be an enzyme, or catalytic domain of an enzyme, whose enzymatic activity mediates negative regulation of intracellular signal transduction. Non-limiting examples of enzymes and enzymatic functions capable of negatively regulating intracellular signal transduction include (1) a kinase or kinase domain whose enzymatic phosphorylation activity mediates negative regulation of intracellular signal transduction, (2) a phosphatase or phosphatase domain whose enzymatic phosphatase activity mediates negative regulation of intracellular signal transduction, and/or (3) a ubiquitin ligase whose enzymatic ubiquitination activity mediates negative regulation of intracellular signal transduction. Enzymatic regulation of signaling (e.g., inhibition intracellular signal transduction cascades) is described in more detail in Pavel Otahal et al. (Biochim Biophys Acta. 2011 February; 1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 June; 14(6): 669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 September; 137(1): 1-19), each of which is incorporated herein by reference for all purposes.
[0089] In some embodiments, the enzymatic inhibitory domain of a chimeric inhibitory receptor of the present disclosure comprises at least a portion of an extracellular domain, a transmembrane domain, and/or an intracellular domain. In some embodiments, the enzymatic inhibitory domain comprises at least a portion of an enzyme, such as a biologically active portion of an enzyme. In some embodiments, the portion of the enzyme comprises an enzyme domain(s), an enzyme fragment(s), or a mutant(s) thereof, such as a kinase domain or a phosphatase domain and mutant thereof. In some embodiments, the portion of the enzyme is a catalytic domain of the enzyme, such as the portion of an enzyme having kinase or phosphatase catalytic activity. In some embodiments, the enzyme domain(s), enzyme fragment(s), or mutants(s) thereof are selected to maximize efficacy and minimize basal inhibition.
[0090] In some embodiments, the enzymatic inhibitory domain comprises one or more modifications that modulate basal inhibition. Examples of modifications include, but are not limited to, truncation mutation(s), amino acid substitution(s), introduction of locations for post-translational modification (examples of which are known to those having skill in the art), and addition of new functional groups. In some embodiments, the enzyme domain(s), enzyme fragment(s), or mutants(s) thereof are selected to maximize efficacy and minimize basal inhibition. In some embodiments, the one or more modifications reduce basal inhibition. In other embodiments, the one or more modifications increase basal inhibition. In a non-limiting illustrative example and without wishing to be bound by theory, deletion of an SH3 domain (e.g., in a CSK enzyme) can minimize constitutive clustering/signaling (i.e., in the absence of ligand binding) and thereby lower the basal level enzymatic inhibitory activity of a chimeric inhibitory receptor.
[0091] In some embodiments, ligand binding between the chimeric inhibitory receptor and its cognate ligand can mediate localization of the chimeric inhibitory receptor to a cellular environment where the enzymatic inhibitory domain is proximal to an intracellular signaling domain or an immune receptor allowing direct inhibition of the signaling activity of the immune receptor. In a non-limiting illustrative example, binding between the chimeric inhibitory receptor expressed on a T cell and its cognate ligand can cause localization of the enzymatic inhibitory domain to be proximal to a TCR or CAR intracellular signaling domain (e.g., localized to a immunological synapse) such that the enzymatic inhibitory domain is capable of negatively regulating T cell signaling and/or activation. In some embodiments, ligand binding between the chimeric inhibitory receptor and its cognate ligand can mediate localization of the chimeric inhibitory receptor to a cellular environment where the enzymatic inhibitory domain is proximal to an immune receptor allowing inhibition of an intracellular signal transduction cascade mediated by the immune receptor. In some embodiments, ligand binding between the chimeric inhibitory receptor and its cognate ligand can mediate spatial clustering of multiple chimeric inhibitory receptors proximal to an immune receptor such that the clustering of the enzymatic inhibitory domains facilitates their inhibitory activity on the immune receptor.
[0092] In some embodiments, the enzyme is selected from CSK, SHP-1, SHP-2, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, ZAP70, and RasGAP.
[0093] In some embodiments, the enzymatic inhibitory domain has a SRC homology 3 (SH3) domain. In some embodiments, the enzymatic inhibitory domain is derived from a protein with a SRC homology 3 (SH3) deletion. In some embodiments, the enzymatic inhibitory domain has a protein tyrosine phosphatase (PTP) domain. In some embodiments, the enzymatic inhibitory domain includes a SRC homology 1 (SH1) domain, a SRC homology 2 (SH2) domain, or an SH1 domain and an SH2 domain.
[0094] In some embodiments, the enzymatic inhibitory domain is derived from a protein with a kinase domain deletion or mutation(s) reducing kinase activity. In some embodiments, the enzymatic inhibitory domain is derived from a protein with a kinase domain deletion or mutation(s) reducing kinase activity generating a dominant negative kinase mutant. In a non-limiting illustrative example and without wishing to be bound by theory, a chimeric inhibitory receptor including enzymatic inhibitory domain having a deletion or mutation of a kinase domain (e.g., in a ZAP70 enzyme) can act as a dominant negative kinase-dead protein and reduce or eliminate an intracellular signaling cascade through competition with the corresponding native wild-type protein that was the source of the enzymatic inhibitory domain.
[0095] In some embodiments, the enzymatic inhibitory domain is derived from CSK. In some embodiments, the enzymatic inhibitory domain derived from CSK is a CSK protein with a SRC homology 3 (SH3) deletion.
[0096] In some embodiments, the enzymatic inhibitory domain is derived from SHP-1. In some embodiments, the enzymatic inhibitory domain derived from SHP-1 has a tyrosine phosphatase (PTP) domain.
[0097] In some embodiments, the enzymatic inhibitory domain is derived from SHP-2. In some embodiments, the enzymatic inhibitory domain is derived from PTEN. In some embodiments, the enzymatic inhibitory domain is derived from CD45. In some embodiments, the enzymatic inhibitory domain is derived from CD148. In some embodiments, the enzymatic inhibitory domain is derived from PTP-MEG1. In some embodiments, the enzymatic inhibitory domain is derived from PTP-PEST. In some embodiments, the enzymatic inhibitory domain is derived from c-CBL. In some embodiments, the enzymatic inhibitory domain is derived from CBL-b. In some embodiments, the enzymatic inhibitory domain is derived from PTPN22. In some embodiments, the enzymatic inhibitory domain is derived from LAR. In some embodiments, the enzymatic inhibitory domain is derived from PTPH1.
[0098] In some embodiments, the enzymatic inhibitory domain is derived from SHIP-1. In some embodiments, the enzymatic inhibitory domain is derived from SHIP-1 has a protein tyrosine phosphatase (PTP) domain.
[0099] In some embodiments, the enzymatic inhibitory domain is derived from ZAP70. In some embodiments, the enzymatic inhibitory domain derived from ZAP70 has a SRC homology 1 (SH1) domain, a SRC homology 2 (SH2) domain, or an SH1 domain and an SH2 domain. In some embodiments, the enzymatic inhibitory domain derived from ZAP70 has a kinase domain deletion. In some embodiments, the enzymatic inhibitory domain derived from ZAP70 has a Tyr492Phe amino acid substitution, a Tyr493Phe amino acid substitution, or a Tyr492Phe amino acid substitution and a Tyr493Phe amino acid substitution.
[0100] In some embodiments, the enzymatic inhibitory domain is derived from RasGAP.
[0101] Exemplary sequences for enzymatic inhibitory domains are shown in Table 1A and Table 1B. In some embodiments, an enzymatic inhibitory domain is any of the amino acid sequences listed in Table 1A. In some embodiments, an enzymatic inhibitory domain has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table 1A. In some embodiments, an enzymatic inhibitory domain is encoded by a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% identical to any of the nucleic acid sequences listed in Table 1B. In some embodiments, an enzymatic inhibitory domain is encoded by a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 1B.
TABLE-US-00002 TABLE 1A Enzymatic Inhibitory Domains Amino Acid Sequences Domain Amino Acid Sequence Csk SAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGR EGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYP GDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADGLCTRLIK PKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVK CIKNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYL RSRGRSVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDF GLTKEASSTQDTGKLPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRI PLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIK THELHL (SEQ ID NO: 1) Csk deltaSH3 VKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKV EHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDE FYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATAQAFLAE ASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGDCL LKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTG KLPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGY KMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELHL (SEQ ID NO: 2) SHP-1 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQN SGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYH GHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIK VMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIE NRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNI LPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFW QMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEY KLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAG PIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQY KFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKED VYENLHTKNKREEKVKKQRSADKEKSKGSLKRK (SEQ ID NO: 3) SHP-1PTP FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD domain YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVE KGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIW HYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDM LMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQF (SEQ ID NO: 4) SHP-2 MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQ NTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSER WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSK VTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQPLNTTRI NAAEIESRVRELSKLAETTDKVKQGFWEEFETLQQQECKLLYSRKEGQRQENKNKN RYKNILPFDHTRVVLHDGDPNEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCL QNTVNDFWRMVFQENSRVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRN VKESAAHDYTLRELKLSKVGQALLQGNTERTVWQYHFRTWPDHGVPSDPGGVLD FLEEVHHKQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQM VRSQRSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIKYSLADQ TSGDQSPLPPCTPTPPCAEMREDSARVYENVGLMQQQKSFR (SEQ ID NO: 5) CD45 YKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFL AEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASY IDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWP SMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHG VPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVD VYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKR DPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELE MSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQ MIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVF ELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHH KSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQ YQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLP EAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS (SEQ ID NO: 6) Grb2 MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEM KPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVL RDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDF DPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV (SEQ ID NO: 7) PTEN MTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLD SKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSE DDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIP SQRRYVYYYSYLLKNHLDYRPVALLFHKMMFETIPMFSGGTCNPQFVVCQLKVKI YSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFI PGPEETSEKVENGSLCDQEIDSICSIERADNDKEYLVLTLTKNDLDKANKDKANRYF SPNFKVKLYFTKTVEEPSNPEASSSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDED QHTQITKV (SEQ ID NO: 8) PTP-MEG1 MTSRFRLPAGRTYNVRASELARDRQHTEVVCNILLLDNTVQAFKVNKHDQGQVLL DVVFKHLDLTEQDYFGLQLADDSTDNPRWLDPNKPIRKQLKRGSPYSLNFRVKFFV SDPNKLQEEYTRYQYFLQIKQDILTGRLPCPSNTAALLASFAVQSELGDYDQSENLS GYLSDYSFIPNQPQDFEKEIAKLHQQHIGLSPAEAEFNYLNTARTLELYGVEFHYAR DQSNNEIMIGVMSGGILIYKNRVRMNTFPWLKIVKISFKCKQFFIQLRKELHESRETL LGFNMVNYRACKNLWKACVEHHTFFRLDRPLPPQKNFFAHYFTLGSKFRYCGRTE VQSVQYGKEKANKDRVFARSPSKPLARKLMDWEVVSRNSISDDRLETQSLPSRSPP GTPNHRNSTFTQEGTRLRPSSVGHLVDHMVHTSPSEVFVNQRSPSSTQANSIVLESSP SQETPGDGKPPALPPKQSKKNSWNQIHYSHSQQDLESHINETFDIPSSPEKPTPNGGIP HDNLVLIRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPADLCVPRLNEGDQV VLINGRDIAEHTHDQVVLFIKASCERHSGELMLLVRPNAVYDVVEEKLENEPDFQYI PEKAPLDSVHQDDHSLRESMIQLAEGLITGTVLTQFDQLYRKKPGMTMSCAKLPQN ISKNRYRDISPYDATRVILKGNEDYINANYINMEIPSSSIINQYIACQGPLPHTCTDFW QMTWEQGSSMVVMLTTQVERGRVKCHQYWPEPTGSSSYGCYQVTCHSEEGNTAY IFRKMTLFNQEKNESRPLTQIQYIAWPDHGVPDDSSDFLDFVCHVRNKRAGKEEPV VVHCSAGIGRTGVLITMETAMCLIECNQPVYPLDIVRTMRDQRAMMIQTPSQYRFV CEAILKVYEEGFVKPLTTSTNK (SEQ ID NO: 9) PTPN22 DQREILQKFLDEAQSKKITKEEFANEFLKLKRQSTKYKADKTYPTTVAEKPKNIKKN RYKDILPYDYSRVELSLITSDEDSSYINANFIKGVYGPKAYIATQGPLSTTLLDFWRM IWEYSVLIIVMACMEYEMGKKKCERYWAEPGEMQLEFGPFSVSCEAEKRKSDYIIR TLKVKFNSETRTIYQFHYKNWPDHDVPSSIDPILELIWDVRCYQEDDSVPICIHCSAG CGRTGVICAIDYTWMLLKDGIIPENFSVFSLIREMRTQRPSLVQTQEQYELVYNAVL ELFKRQMDVIRDKHSGTESQAKHCIPEKNHTLQADSYSPNLPKSTTKAAKMMNQQ RTKMEIKESSSFDFRTSEISAKEELVLHPAKSSTSFDFLELNYSFDKNADTTMKWQT KAFPIVGEPLQKHQSLDLGSLLFEGCSNSKPVNAAGRYFNSKVPITRTKSTPFELIQQ RETKEVDSKENFSYLESQPHDSCFVEMQAQKVMHVSSAELNYSLPYDSKHQIRNAS NVKHHDSSALGVYSYIPLVENPYFSSWPPSGTSSKMSLDLPEKQDGTVFPSSLLPTSS TSLFSYYNSHDSLSLNSPTNISSLLNQESAVLATAPRIDDEIPPPLPVRTPESFIVVEEA GEFSPNVPKSLSSAVKVKIGTSLEWGGTSEPKKFDDSVILRPSKSVKLRSPKSELHQD RSSPPPPLPERTLESFFLADEDCMQAQSIETYSTSYPDTMENSTSSKQTLKTPGKSFTR SKSLKILRNMKKSICNSCPPNKPAESVQSNNSSSFLNFGFANRFSKPKGPRNPPPTWN I(SEQ ID NO: 10) Zap70 SH2 1, MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRF SH2 2 domains HHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPG VFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSL TREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPE GTKFDTLWQLVEYLKLKADGLIYCLKEAC (SEQ ID NO: 11) Zap70 delta MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRF kinase HHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPG VFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSL TREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPE GTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQ RRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNL (SEQ ID NO: 12) Zap70 Y492F MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRF Y493F HHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPG VFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSL TREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPE GTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQ RRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLI ADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLD NPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGM KYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSFFTARSAGKWPLK WYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRM ECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKA EAACA (SEQ ID NO: 13) SHIP-1 PTP DMITIFIGTWNMGNAPPPKKITSWFLSKGQGKTRDDSADYIPHDIYVIGTQEDPLSEK domain EWLEILKHSLQEITSVTFKTVAIHTLWNIRIVVLAKPEHENRISHICTDNVKTGIANTL GNKGAVGVSFMFNGTSLGFVNSHLTSGSEKKLRRNQNYMNILRFLALGDKKLSPFN ITHRFTHLFWFGDLNYRVDLPTWEAETIIQKIKQQQYADLLSHDQLLTERREQKVFL HFEEEEITFAPTYRFERLTRDKYAYTKQKATGMKYNLPSWCDRVLWKSYPLVHVV CQSYGSTSDIMTSDHSPVFATFEAGVTSQFVS (SEQ ID NO: 14)
TABLE-US-00003 TABLE 1B Enzymatic Inhibitory Domains Nucleic Acid Sequences Domain Nucleic Acid Sequence Csk AGTGCCATTCAGGCTGCATGGCCGTCAGGTACGGAGTGTATTGCTAAATACAAT TTTCACGGGACTGCTGAACAGGACCTTCCTTTTTGCAAAGGGGACGTCTTGACT ATCGTAGCTGTGACGAAAGACCCGAACTGGTACAAAGCTAAGAATAAGGTCGG CCGGGAGGGTATAATTCCCGCAAATTACGTGCAGAAGCGAGAAGGTGTTAAAG CTGGAACAAAGTTGTCACTCATGCCGTGGTTTCATGGAAAAATTACCAGAGAAC AAGCGGAGCGGCTGTTGTACCCGCCGGAAACCGGCCTTTTCTTGGTTAGGGAAA GCACCAATTACCCTGGGGACTACACTCTTTGTGTTTCATGCGATGGGAAGGTAG AACATTACCGCATCATGTATCATGCCAGTAAGCTTTCCATAGACGAAGAGGTGT ACTTCGAGAACCTGATGCAACTGGTGGAGCACTACACATCCGATGCGGACGGAT TGTGCACGCGATTGATAAAACCAAAGGTAATGGAAGGCACCGTGGCAGCTCAG GATGAGTTTTACCGAAGCGGTTGGGCACTGAACATGAAAGAACTTAAACTGCTT CAGACAATTGGGAAAGGAGAATTTGGCGACGTGATGCTGGGTGATTATAGGGG TAACAAGGTCGCGGTGAAGTGCATTAAGAACGATGCCACGGCCCAGGCCTTTCT GGCAGAAGCTTCAGTTATGACTCAACTCCGCCATTCTAATCTGGTCCAATTGCTT GGAGTGATCGTCGAAGAGAAGGGAGGCCTCTACATAGTAACGGAGTATATGGC TAAAGGTTCTCTCGTCGATTATCTCCGCTCACGCGGGCGCAGCGTTCTCGGGGG AGATTGCCTCCTGAAATTCAGTCTGGACGTCTGCGAGGCTATGGAGTACCTCGA AGGAAACAATTTTGTCCACCGGGACTTGGCTGCAAGGAACGTCCTGGTAAGCGA AGATAACGTGGCTAAGGTGTCCGACTTCGGGCTCACGAAAGAAGCAAGTAGTA CTCAAGACACGGGCAAACTCCCAGTTAAATGGACCGCACCGGAGGCACTCAGG GAAAAAAAATTTTCTACCAAGAGTGACGTATGGTCATTCGGCATCCTCTTGTGG GAAATATATAGTTTTGGGCGCGTTCCTTACCCAAGAATCCCCCTGAAGGATGTC GTGCCGAGAGTGGAAAAGGGGTACAAGATGGATGCTCCGGACGGATGCCCCCC AGCAGTATATGAGGTAATGAAAAATTGCTGGCATCTCGATGCAGCGATGCGGCC GTCCTTTCTTCAGCTCCGGGAGCAACTGGAACACATAAAGACGCACGAACTTCA TCTT (SEQ ID NO: 15) Csk de1taSH3 GTTAAAGCTGGAACAAAGTTGTCACTCATGCCGTGGTTTCATGGAAAAATTACC AGAGAACAAGCGGAGCGGCTGTTGTACCCGCCGGAAACCGGCCTTTTCTTGGTT AGGGAAAGCACCAATTACCCTGGGGACTACACTCTTTGTGTTTCATGCGATGGG AAGGTAGAACATTACCGCATCATGTATCATGCCAGTAAGCTTTCCATAGACGAA GAGGTGTACTTCGAGAACCTGATGCAACTGGTGGAGCACTACACATCCGATGCG GACGGATTGTGCACGCGATTGATAAAACCAAAGGTAATGGAAGGCACCGTGGC AGCTCAGGATGAGTTTTACCGAAGCGGTTGGGCACTGAACATGAAAGAACTTAA ACTGCTTCAGACAATTGGGAAAGGAGAATTTGGCGACGTGATGCTGGGTGATTA TAGGGGTAACAAGGTCGCGGTGAAGTGCATTAAGAACGATGCCACGGCCCAGG CCTTTCTGGCAGAAGCTTCAGTTATGACTCAACTCCGCCATTCTAATCTGGTCCA ATTGCTTGGAGTGATCGTCGAAGAGAAGGGAGGCCTCTACATAGTAACGGAGT ATATGGCTAAAGGTTCTCTCGTCGATTATCTCCGCTCACGCGGGCGCAGCGTTCT CGGGGGAGATTGCCTCCTGAAATTCAGTCTGGACGTCTGCGAGGCTATGGAGTA CCTCGAAGGAAACAATTTTGTCCACCGGGACTTGGCTGCAAGGAACGTCCTGGT AAGCGAAGATAACGTGGCTAAGGTGTCCGACTTCGGGCTCACGAAAGAAGCAA GTAGTACTCAAGACACGGGCAAACTCCCAGTTAAATGGACCGCACCGGAGGCA CTCAGGGAAAAAAAATTTTCTACCAAGAGTGACGTATGGTCATTCGGCATCCTC TTGTGGGAAATATATAGTTTTGGGCGCGTTCCTTACCCAAGAATCCCCCTGAAG GATGTCGTGCCGAGAGTGGAAAAGGGGTACAAGATGGATGCTCCGGACGGATG CCCCCCAGCAGTATATGAGGTAATGAAAAATTGCTGGCATCTCGATGCAGCGAT GCGGCCGTCCTTTCTTCAGCTCCGGGAGCAACTGGAACACATAAAGACGCACGA ACTTCATCTT (SEQ ID NO: 16) SHP-1 ATGGTTCGATGGTTCCACAGAGATCTGAGCGGCCTGGATGCCGAGACTCTGCTT AAAGGACGGGGCGTGCACGGCAGCTTTCTGGCTAGACCCAGCAGAAAGAACCA GGGCGACTTCAGCCTGTCCGTCAGAGTGGGCGATCAAGTGACACACATCAGAAT CCAGAACTCCGGCGACTTCTACGACCTGTACGGCGGCGAGAAGTTCGCCACACT GACAGAGCTGGTGGAATATTACACCCAGCAGCAAGGCGTGCTGCAGGACAGAG ATGGCACCATCATCCACCTGAAGTACCCTCTGAACTGCAGCGACCCCACCAGCG AGAGATGGTATCACGGACACATGTCTGGCGGCCAGGCTGAGACACTGCTTCAGG CTAAAGGCGAGCCCTGGACCTTTCTCGTGCGGGAATCTCTGAGTCAGCCCGGCG ATTTTGTGCTGAGCGTGCTGTCCGATCAGCCCAAAGCTGGACCAGGCTCTCCAC TGAGAGTGACCCATATCAAAGTGATGTGCGAAGGCGGACGGTACACCGTCGGT GGCCTGGAAACATTCGATAGCCTGACCGACCTGGTCGAGCACTTCAAGAAAACC GGCATCGAGGAAGCCAGCGGCGCCTTCGTTTATCTGAGACAGCCCTACTACGCC ACAAGAGTGAACGCCGCCGACATCGAGAACAGAGTGCTGGAACTGAACAAGAA GCAAGAGAGCGAGGATACCGCCAAGGCCGGCTTCTGGGAAGAGTTCGAGTCCC TGCAGAAACAAGAAGTGAAGAACCTGCACCAGCGGCTGGAAGGACAGAGGCCT GAGAACAAGGGCAAGAACCGGTACAAGAACATCCTGCCATTCGACCACTCCAG AGTGATCCTGCAGGGAAGAGACAGCAACATCCCCGGCTCCGACTACATCAACG CCAATTACATCAAGAACCAGCTGCTGGGCCCCGACGAGAACGCCAAGACATAT ATCGCCTCTCAGGGCTGCCTGGAAGCCACCGTGAACGACTTTTGGCAGATGGCC TGGCAAGAGAACAGCCGCGTGATCGTGATGACCACCAGAGAGGTGGAAAAAGG CCGGAACAAATGCGTGCCCTACTGGCCCGAAGTGGGCATGCAAAGAGCCTACG GACCTTACAGCGTGACCAACTGCGGCGAGCACGATACCACCGAGTACAAGCTG AGAACCCTCCAGGTGTCCCCTCTGGACAACGGCGACCTGATCAGAGAGATCTGG CACTACCAGTACCTGTCCTGGCCTGATCACGGCGTGCCATCTGAACCTGGTGGC GTGCTGAGTTTCCTGGACCAGATCAACCAGAGACAAGAGAGCCTGCCTCACGCT GGCCCTATCATCGTGCACTGTTCTGCCGGCATCGGCAGAACCGGCACAATCATC GTGATCGACATGCTGATGGAAAACATCAGCACCAAGGGCCTCGACTGCGACATC GACATCCAGAAAACCATCCAGATGGTTCGAGCCCAGCGGAGCGGAATGGTGCA GACAGAAGCCCAGTACAAGTTCATCTACGTGGCAATCGCCCAGTTCATCGAAAC CACCAAGAAAAAGCTGGAAGTGCTGCAGTCCCAGAAGGGCCAAGAAAGCGAGT ACGGCAACATCACATACCCTCCAGCCATGAAGAACGCCCACGCCAAAGCCAGC AGAACCTCCAGCAAGCACAAAGAGGACGTCTACGAGAATCTGCACACAAAGAA CAAGCGCGAGGAAAAAGTGAAAAAGCAGCGCAGCGCCGACAAAGAGAAGTCC AAGGGCAGCCTGAAGAGAAAG (SEQ ID NO: 17) SHP-1 PTP TTCTGGGAAGAGTTCGAGTCCCTGCAGAAACAAGAAGTGAAGAACCTGCACCA domain GCGGCTGGAAGGACAGAGGCCTGAGAACAAGGGCAAGAACCGGTACAAGAAC ATCCTGCCATTCGACCACTCCAGAGTGATCCTGCAGGGAAGAGACAGCAACATC CCCGGCTCCGACTACATCAACGCCAATTACATCAAGAACCAGCTGCTGGGCCCC GACGAGAACGCCAAGACATATATCGCCTCTCAGGGCTGCCTGGAAGCCACCGTG AACGACTTTTGGCAGATGGCCTGGCAAGAGAACAGCCGCGTGATCGTGATGACC ACCAGAGAGGTGGAAAAAGGCCGGAACAAATGCGTGCCCTACTGGCCCGAAGT GGGCATGCAAAGAGCCTACGGACCTTACAGCGTGACCAACTGCGGCGAGCACG ATACCACCGAGTACAAGCTGAGAACCCTCCAGGTGTCCCCTCTGGACAACGGCG ACCTGATCAGAGAGATCTGGCACTACCAGTACCTGTCCTGGCCTGATCACGGCG TGCCATCTGAACCTGGTGGCGTGCTGAGTTTCCTGGACCAGATCAACCAGAGAC AAGAGAGCCTGCCTCACGCTGGCCCTATCATCGTGCACTGTTCTGCCGGCATCG GCAGAACCGGCACAATCATCGTGATCGACATGCTGATGGAAAACATCAGCACC AAGGGCCTCGACTGCGACATCGACATCCAGAAAACCATCCAGATGGTTCGAGCC CAGCGGAGCGGAATGGTGCAGACAGAAGCCCAGTACAAGTTCATCTACGTGGC AATCGCCCAGTTC (SEQ ID NO: 18) SHP-2 ATGACAAGCAGACGGTGGTTTCACCCCAACATCACCGGCGTGGAAGCTGAGAA TCTGCTGCTGACAAGAGGCGTGGACGGCAGCTTTCTGGCTAGACCCAGCAAGTC CAATCCTGGCGACTTCACACTGAGCGTGCGGAGAAATGGCGCCGTGACACACAT CAAGATCCAGAACACCGGCGACTACTACGACCTGTACGGCGGCGAGAAGTTTG CCACACTGGCAGAGCTGGTGCAGTACTACATGGAACACCACGGCCAGCTGAAA GAAAAGAACGGCGACGTGATCGAGCTGAAGTACCCTCTGAACTGCGCCGATCCT ACCAGCGAGAGATGGTTTCACGGCCACCTGTCTGGCAAAGAGGCCGAGAAGCT GCTGACCGAGAAGGGCAAGCACGGAAGCTTTCTCGTGCGCGAGTCTCAGTCTCA CCCCGGCGATTTTGTGCTGTCTGTGCGGACAGGGGACGACAAGGGCGAGAGCA ATGACGGCAAGAGCAAAGTGACCCACGTGATGATCCGGTGCCAAGAGCTGAAA TACGACGTCGGCGGAGGGGAGAGATTCGACTCTCTGACCGATCTGGTGGAACAC TACAAGAAAAACCCCATGGTGGAAACCCTGGGCACCGTGCTGCAGCTGAAGCA GCCACTGAACACCACCAGAATCAACGCCGCCGAGATCGAGAGCAGAGTGCGGG AACTGTCTAAGCTGGCCGAGACTACCGACAAAGTGAAGCAAGGCTTCTGGGAA GAGTTCGAGACACTGCAGCAGCAAGAGTGCAAGCTGCTGTACTCCCGGAAAGA GGGCCAGAGACAAGAGAACAAGAACAAAAACCGGTACAAGAACATCCTGCCGT TCGATCACACCAGAGTGGTGCTGCACGACGGCGATCCTAATGAGCCCGTGTCCG ACTACATCAACGCCAACATCATCATGCCCGAGTTTGAGACAAAGTGCAACAATA GCAAGCCCAAGAAGTCCTATATCGCCACACAGGGCTGCCTGCAGAATACCGTGA ACGACTTTTGGCGGATGGTGTTTCAAGAGAACTCCCGCGTGATCGTGATGACCA CCAAAGAGGTGGAACGGGGCAAGTCTAAGTGCGTGAAGTACTGGCCCGACGAG TACGCCCTGAAAGAATACGGCGTGATGAGAGTGCGGAACGTGAAAGAGAGCGC CGCTCACGATTACACCCTGAGAGAGCTGAAGCTGAGCAAAGTCGGACAGGCCC TGCTGCAGGGAAACACCGAAAGAACCGTGTGGCAGTACCACTTCCGGACCTGG CCAGATCATGGCGTGCCATCTGATCCTGGCGGCGTGCTGGATTTCCTGGAAGAG GTGCACCACAAGCAAGAGTCCATCATGGACGCCGGACCAGTGGTGGTGCACTGT TCTGCCGGAATCGGAAGAACCGGCACCTTCATCGTGATCGACATCCTGATTGAC ATCATCCGCGAGAAAGGCGTCGACTGCGATATCGACGTGCCCAAGACCATCCAG ATGGTTCGAAGCCAGAGAAGCGGCATGGTGCAGACAGAGGCCCAGTACCGGTT CATCTACATGGCCGTGCAGCATTACATCGAAACCCTGCAGCGGCGGATCGAGGA AGAACAGAAGTCCAAGAGAAAGGGCCACGAGTACACCAACATCAAGTACAGCC TGGCCGACCAGACCAGCGGCGATCAATCTCCTCTGCCTCCTTGCACACCCACAC CTCCATGTGCCGAGATGCGGGAAGATAGCGCCAGGGTGTACGAGAACGTGGGC CTGATGCAACAGCAGAAGTCCTTCCGG (SEQ ID NO: 19) CD45 TACAAGATCTACGACCTGCACAAGAAGCGGAGCTGCAATCTGGACGAGCAGCA AGAACTGGTGGAACGGGACGACGAGAAGCAGCTGATGAACGTGGAACCCATCC ACGCCGACATCCTGCTGGAAACCTACAAGCGGAAGATCGCCGACGAGGGCAGA CTGTTCCTGGCCGAGTTTCAGAGCATCCCCAGAGTGTTCAGCAAGTTCCCCATCA AAGAGGCCAGAAAGCCCTTCAACCAGAACAAGAACCGCTACGTGGACATTCTG CCCTACGACTACAATCGCGTGGAACTGAGCGAGATCAATGGCGACGCCGGCAG CAACTACATCAACGCCAGCTACATCGACGGCTTCAAAGAGCCCCGGAAGTATAT CGCCGCTCAGGGCCCTAGAGATGAGACAGTGGACGACTTCTGGCGCATGATTTG GGAGCAGAAAGCCACCGTGATCGTGATGGTTACCAGATGCGAAGAGGGCAACA GAAACAAGTGCGCCGAGTACTGGCCCAGCATGGAAGAAGGCACAAGAGCCTTT GGCGACGTGGTGGTCAAGATCAATCAGCACAAGCGGTGCCCCGACTACATCATC CAGAAACTGAACATCGTGAACAAGAAAGAGAAGGCCACCGGACGGGAAGTGA CCCACATCCAGTTTACCAGCTGGCCCGATCATGGCGTGCCCGAGGATCCACATC TGCTGCTCAAGCTGCGGAGAAGAGTGAACGCCTTCAGCAACTTCTTCAGCGGCC CCATCGTGGTGCACTGTTCTGCAGGCGTTGGAAGAACCGGCACCTACATCGGAA TCGACGCCATGCTGGAAGGACTGGAAGCCGAGAACAAGGTGGACGTGTACGGC TACGTGGTCAAGCTGAGAAGGCAGCGGTGTCTGATGGTGCAGGTTGAGGCCCA GTACATCCTGATCCATCAGGCCCTGGTCGAGTACAACCAGTTCGGCGAGACAGA AGTGAACCTGAGCGAGCTGCACCCCTATCTGCACAACATGAAGAAGCGGGACC CTCCAAGCGAGCCCTCTCCACTGGAAGCTGAGTTCCAGAGACTGCCCAGCTACA GAAGCTGGCGGACACAGCACATCGGCAATCAAGAGGAAAACAAGAGCAAGAA CCGGAACAGCAACGTGATCCCGTACGATTACAACAGAGTGCCCCTGAAGCACG AACTCGAGATGAGCAAAGAGAGCGAGCACGACAGCGACGAGTCCAGCGACGAT GATAGCGACAGCGAGGAACCCAGCAAGTATATCAATGCCTCCTTCATCATGAGC TATTGGAAGCCCGAAGTGATGATTGCCGCACAGGGACCCCTGAAAGAGACAAT CGGCGACTTTTGGCAGATGATCTTCCAGCGGAAAGTGAAAGTGATCGTCATGCT GACCGAGCTGAAACACGGCGACCAAGAGATCTGCGCCCAGTATTGGGGAGAGG GAAAGCAGACCTACGGCGACATTGAGGTGGACCTGAAGGACACCGACAAGAGC AGCACCTACACACTGCGGGTGTTCGAGCTGAGACACTCCAAGAGAAAGGACAG CCGGACCGTGTACCAGTACCAGTATACCAATTGGAGCGTGGAACAGCTGCCTGC CGAGCCTAAAGAACTGATCAGCATGATCCAGGTCGTGAAGCAGAAGCTGCCTC AGAAGAACAGCAGCGAGGGAAACAAGCACCACAAGTCTACCCCTCTGCTGATC CACTGCAGAGATGGCTCTCAGCAGACCGGCATCTTCTGCGCCCTGCTGAATCTC CTGGAAAGCGCCGAGACAGAGGAAGTGGTGGACATCTTCCAGGTGGTCAAAGC CCTGCGGAAGGCCAGACCTGGCATGGTGTCTACCTTCGAGCAGTATCAGTTCCT GTACGACGTGATCGCCAGCACATACCCCGCTCAGAACGGCCAAGTGAAGAAGA ACAACCACCAAGAGGACAAGATCGAGTTCGACAACGAGGTGGACAAAGTGAAG CAGGACGCCAACTGCGTGAACCCTCTGGGAGCCCCAGAAAAGCTGCCTGAGGC CAAAGAACAGGCCGAGGGCTCTGAGCCAACATCTGGAACAGAGGGACCTGAGC ACAGCGTGAACGGACCTGCTAGCCCCGCTCTGAATCAGGGCTCT (SEQ ID NO: 20) Grb2 ATGGAAGCCATTGCCAAATACGACTTCAAGGCCACCGCCGACGACGAGCTGAG CTTCAAGAGAGGCGACATCCTGAAGGTGCTGAACGAGGAATGCGACCAGAACT GGTACAAGGCCGAGCTGAACGGCAAGGACGGCTTCATCCCCAAGAACTACATC GAGATGAAGCCCCATCCATGGTTCTTCGGCAAGATCCCCAGAGCCAAGGCCGAA GAGATGCTGAGCAAGCAGAGACACGACGGCGCCTTTCTGATCCGGGAATCTGA ATCTGCCCCTGGCGACTTCAGCCTGTCCGTGAAGTTCGGCAACGACGTGCAGCA CTTCAAGGTCCTGAGAGATGGCGCCGGAAAGTACTTCCTGTGGGTCGTGAAGTT TAACAGCCTGAACGAGCTGGTGGACTACCACAGATCTACCAGCGTGTCCCGGAA CCAGCAGATCTTCCTGCGGGACATCGAACAGGTTCCCCAGCAACCTACCTACGT GCAGGCCCTGTTCGACTTCGACCCTCAAGAGGATGGCGAGCTGGGCTTTAGACG GGGCGATTTCATCCACGTGATGGACAATAGCGACCCCAACTGGTGGAAGGGCG CCTGTCATGGACAGACCGGCATGTTCCCCAGAAACTACGTGACCCCTGTGAACC GGAACGTG (SEQ ID NO: 21) PTEN ATGACAGCCATCATCAAAGAAATCGTGTCCCGGAACAAGCGGCGCTACCAAGA GGATGGCTTCGACCTGGACCTGACCTACATCTACCCCAACATCATTGCCATGGG CTTCCCCGCCGAAAGACTGGAAGGCGTGTACAGAAACAACATCGACGATGTCGT GCGGTTCCTGGACAGCAAGCACAAGAACCACTACAAGATCTACAACCTGTGCGC CGAGCGGCACTACGATACCGCCAAGTTCAACTGCAGAGTGGCTCAGTACCCCTT CGAGGACCACAATCCTCCACAGCTGGAACTGATCAAGCCCTTCTGCGAGGACCT GGATCAGTGGCTGAGCGAGGACGATAATCACGTGGCCGCCATTCACTGCAAGG CCGGCAAGGGAAGAACCGGCGTGATGATCTGTGCCTACCTGCTGCACCGGGGC AAGTTTCTGAAAGCCCAAGAGGCCCTGGACTTCTACGGCGAAGTGCGGACCAG AGACAAGAAAGGCGTGACAATCCCCAGCCAGCGGAGATACGTGTACTACTACA GCTATCTGCTGAAGAACCACCTGGACTACAGACCCGTGGCACTGCTGTTCCACA AGATGATGTTCGAGACTATCCCCATGTTCAGCGGCGGCACATGCAACCCTCAGT TCGTCGTGTGCCAGCTGAAAGTGAAGATCTACTCCAGCAACAGCGGCCCCACCA GACGCGAGGACAAGTTCATGTACTTCGAGTTCCCTCAGCCTCTGCCTGTGTGCG GCGACATCAAGGTGGAATTCTTCCACAAGCAGAACAAGATGCTGAAAAAGGAC AAGATGTTCCACTTCTGGGTCAACACCTTCTTCATCCCCGGACCTGAAGAGACA AGCGAGAAGGTGGAAAACGGCAGCCTGTGCGACCAAGAGATCGACAGCATCTG CAGCATCGAGCGGGCCGACAACGACAAAGAATACCTGGTGCTGACCCTGACCA AGAACGATCTGGACAAGGCCAACAAGGATAAGGCCAACCGGTACTTCAGCCCC AACTTCAAAGTGAAACTGTACTTCACCAAGACCGTCGAGGAACCCAGCAATCCT GAGGCCAGCTCTAGCACATCTGTGACCCCTGACGTGTCCGACAATGAGCCCGAC CACTACAGATACAGCGACACCACCGACAGCGACCCCGAGAACGAGCCTTTCGA TGAGGATCAGCACACACAGATCACCAAAGTG (SEQ ID NO: 22) PTP-MEG1 ATGACAAGCAGATTCAGACTGCCAGCCGGCAGAACCTACAATGTGCGGGCTTCT GAGCTGGCCAGAGACAGACAGCACACCGAGGTCGTGTGCAACATCCTGCTGCTC GACAATACCGTGCAGGCCTTCAAAGTGAACAAGCACGACCAGGGCCAAGTCCT GCTGGACGTGGTGTTCAAGCACCTGGATCTGACCGAGCAGGACTACTTCGGACT GCAGCTGGCCGACGACAGCACCGACAATCCTAGATGGCTGGACCCCAACAAGC CCATCCGGAAGCAGCTGAAGAGAGGCTCCCCTTACTCTCTGAACTTCCGGGTCA AGTTCTTCGTGTCCGATCCTAACAAACTGCAAGAAGAGTATACCCGCTACCAGT ACTTCCTGCAGATCAAGCAGGACATCCTGACCGGCAGACTGCCCTGTCCTTCTA ATACTGCCGCTCTGCTGGCCTCCTTTGCCGTGCAATCTGAACTGGGCGACTACGA CCAGAGCGAGAACCTGAGCGGCTACCTGAGCGATTACAGCTTCATCCCCAACCA GCCTCAGGACTTCGAGAAAGAGATCGCCAAGCTGCATCAGCAGCACATCGGAC TGTCTCCAGCCGAGGCCGAGTTCAACTACCTGAACACCGCCAGGACACTGGAAC TGTACGGCGTGGAATTTCACTACGCCCGGGACCAGAGCAACAACGAGATCATG ATTGGCGTGATGAGCGGCGGCATCCTGATCTACAAGAACAGAGTGCGGATGAA CACATTCCCCTGGCTGAAGATCGTGAAGATCAGCTTCAAGTGCAAGCAGTTCTT CATCCAGCTGCGGAAAGAGCTGCACGAGAGCAGAGAGACACTGCTGGGCTTCA ACATGGTCAACTACCGGGCCTGCAAGAACCTGTGGAAGGCCTGTGTGGAACACC ACACCTTTTTCCGGCTGGACCGACCTCTGCCTCCTCAGAAGAATTTCTTCGCCCA CTACTTCACCCTGGGCAGCAAGTTCAGATACTGCGGCAGAACAGAGGTGCAGTC CGTGCAGTACGGCAAAGAGAAAGCCAACAAGGACCGGGTGTTCGCCAGATCTC CTAGCAAGCCACTGGCCAGAAAGCTGATGGACTGGGAAGTCGTGTCCCGGAAC AGCATCAGCGACGACAGACTGGAAACCCAGAGCCTGCCTAGCAGAAGCCCTCC TGGCACACCCAACCACAGAAACAGCACCTTCACACAAGAGGGCACAAGACTGA GGCCTAGCTCTGTGGGACACCTGGTGGATCACATGGTGCACACAAGCCCCAGCG AGGTGTTCGTGAACCAGAGAAGCCCTAGCTCTACCCAGGCCAACAGCATCGTGC TGGAAAGCAGCCCCAGCCAAGAAACACCAGGCGACGGAAAACCTCCTGCTCTG CCACCTAAGCAGAGCAAGAAGAACAGCTGGAACCAGATCCACTACAGCCACAG CCAGCAGGATCTGGAAAGCCACATCAACGAGACATTCGACATCCCTAGCAGCCC
CGAGAAGCCCACACCTAATGGCGGAATCCCTCACGACAACCTGGTGCTGATCCG GATGAAGCCCGACGAGAATGGCAGATTCGGCTTCAACGTGAAAGGCGGCTACG ATCAGAAAATGCCCGTGATCGTGTCCAGAGTGGCCCCTGGAACTCCTGCCGATC TGTGTGTGCCCAGACTGAACGAAGGCGACCAGGTCGTGCTGATCAACGGCAGA GATATCGCCGAGCACACCCACGATCAGGTGGTGCTGTTCATTAAGGCCTCTTGC GAGAGACACAGCGGCGAACTGATGCTGCTCGTGCGGCCTAATGCCGTGTACGAC GTGGTGGAAGAGAAACTGGAAAACGAGCCCGACTTCCAGTACATCCCTGAGAA GGCCCCACTGGACAGCGTGCACCAGGATGATCATAGCCTGCGCGAGAGCATGA TCCAGCTGGCAGAGGGACTGATCACCGGCACAGTGCTGACCCAGTTCGACCAGC TGTACCGGAAGAAACCTGGCATGACCATGTCCTGCGCCAAACTGCCTCAGAACA TCAGCAAGAACCGGTACAGAGACATCAGCCCCTACGATGCCACCAGAGTGATC CTGAAGGGCAACGAGGACTACATCAACGCCAATTACATCAACATGGAAATCCC CAGCTCCAGCATCATCAACCAGTATATCGCCTGTCAGGGCCCACTGCCTCACAC CTGTACCGACTTTTGGCAGATGACCTGGGAGCAGGGCAGCAGCATGGTGGTCAT GCTGACAACCCAGGTGGAACGGGGCAGAGTGAAGTGCCACCAGTATTGGCCTG AGCCTACCGGCAGCAGCTCCTACGGCTGTTACCAAGTGACCTGCCACAGCGAAG AGGGCAACACCGCCTACATCTTCAGAAAGATGACCCTGTTCAATCAAGAGAAG AACGAGAGCCGGCCTCTGACACAGATCCAGTATATTGCTTGGCCCGACCACGGC GTGCCCGACGATAGTTCTGACTTCCTGGACTTCGTGTGCCACGTGCGCAACAAA CGCGCCGGAAAAGAGGAACCTGTCGTCGTCCACTGTAGCGCCGGCATTGGAAG AACCGGCGTGCTGATTACCATGGAAACAGCCATGTGCCTGATCGAGTGCAATCA GCCCGTGTATCCCCTGGACATCGTGCGGACCATGAGAGATCAGCGGGCCATGAT GATCCAGACACCTAGCCAGTACAGATTCGTGTGCGAGGCCATTCTGAAGGTGTA CGAAGAGGGATTCGTGAAGCCCCTGACCACCTCCACCAACAAG (SEQ ID NO: 23) PTPN22 GATCAGAGAGAGATCCTGCAGAAGTTCCTGGACGAGGCCCAGAGCAAGAAGAT CACCAAAGAGGAATTCGCCAACGAGTTCCTGAAACTGAAGCGGCAGAGCACCA AGTACAAGGCCGACAAGACATACCCCACCACCGTGGCCGAGAAGCCCAAGAAC ATCAAGAAGAACCGGTACAAGGACATCCTGCCATACGACTACTCCAGAGTGGA ACTGAGCCTGATCACCAGCGACGAGGACAGCAGCTACATCAACGCCAACTTCAT CAAGGGCGTGTACGGCCCCAAGGCCTATATCGCAACACAGGGCCCTCTGTCTAC AACCCTGCTGGACTTCTGGCGCATGATCTGGGAGTACAGCGTGCTGATCATCGT GATGGCCTGCATGGAATACGAGATGGGCAAGAAGAAGTGCGAGCGGTACTGGG CCGAACCTGGCGAAATGCAGCTGGAATTCGGCCCCTTTTCCGTGTCCTGCGAAG CCGAGAAGAGAAAGTCCGACTACATCATCAGGACCCTGAAAGTGAAGTTCAAC AGCGAAACCCGGACCATCTACCAGTTTCACTACAAGAACTGGCCCGACCACGAC GTGCCAAGCAGCATCGATCCTATCCTGGAACTGATTTGGGACGTGCGGTGCTAC CAAGAGGACGACAGCGTGCCAATCTGCATCCACTGTTCTGCCGGCTGCGGAAGA ACAGGCGTCATCTGCGCCATCGACTACACCTGGATGCTGCTGAAGGACGGCATC ATCCCCGAGAACTTCAGCGTGTTCAGCCTGATCCGCGAGATGAGAACCCAGAGG CCTAGCCTGGTGCAGACCCAAGAGCAGTACGAACTGGTGTACAACGCCGTGCTG GAACTGTTCAAGAGACAGATGGACGTGATCCGGGACAAGCACAGCGGCACAGA GTCTCAGGCCAAACACTGCATCCCTGAGAAGAATCACACCCTGCAGGCCGACAG CTACAGCCCCAATCTGCCTAAGAGCACCACCAAGGCCGCCAAAATGATGAACC AGCAGCGGACAAAGATGGAAATCAAAGAGAGCAGCTCCTTCGACTTCCGGACC AGCGAGATCAGCGCCAAAGAAGAACTGGTTCTGCACCCCGCCAAGTCCTCTACC AGCTTCGACTTTCTCGAGCTGAACTACAGCTTCGATAAGAACGCCGACACCACC ATGAAGTGGCAGACCAAGGCCTTTCCTATCGTGGGCGAGCCTCTGCAGAAACAC CAGAGCCTGGATCTGGGCTCCCTGCTGTTTGAGGGCTGCAGCAATAGCAAGCCC GTGAACGCCGCTGGCCGGTACTTTAATAGCAAGGTGCCCATCACCAGGACCAAG AGCACCCCTTTCGAGCTGATCCAGCAGCGCGAGACAAAAGAGGTGGACAGCAA AGAGAACTTCTCCTACCTGGAAAGCCAGCCTCACGACAGCTGCTTCGTGGAAAT GCAGGCCCAGAAAGTGATGCACGTGTCCAGCGCCGAGCTGAATTACTCTCTGCC CTACGACAGCAAGCACCAGATCCGGAACGCCAGCAACGTGAAGCACCACGATA GCTCTGCCCTGGGAGTGTACAGCTACATTCCCCTGGTGGAAAACCCCTACTTCA GCTCCTGGCCACCTAGCGGCACAAGCAGCAAGATGTCTCTGGATCTGCCCGAGA AGCAGGACGGCACAGTGTTCCCATCTAGCCTGCTGCCTACCAGCAGCACCAGCC TGTTCAGCTACTACAATAGCCACGACTCTCTGTCCCTGAACAGCCCCACCAACA TCTCCAGCCTGCTGAATCAAGAAAGCGCTGTGCTGGCCACCGCTCCAAGAATCG ACGATGAGATCCCTCCTCCTCTGCCTGTGCGGACCCCTGAGTCTTTCATCGTGGT GGAAGAGGCCGGCGAGTTCAGCCCTAATGTGCCCAAATCTCTGAGCAGCGCCGT GAAAGTCAAGATCGGCACCTCTCTGGAATGGGGCGGCACATCCGAGCCTAAGA AATTCGACGACTCCGTGATCCTGAGGCCAAGCAAGAGCGTGAAGCTGAGAAGC CCCAAGTCCGAGCTGCATCAGGACAGATCTAGCCCTCCTCCACCACTGCCTGAG AGAACCCTCGAGTCATTCTTCCTGGCCGACGAGGATTGCATGCAGGCACAGAGC ATCGAGACATACAGCACAAGCTACCCCGACACCATGGAAAACAGCACCTCCAG CAAGCAGACACTGAAAACCCCAGGCAAGAGCTTCACCCGGTCCAAGAGCCTGA AGATCCTGCGGAACATGAAGAAGTCCATCTGCAACAGCTGCCCTCCAAACAAGC CTGCCGAGAGCGTGCAGTCCAACAATAGCAGCAGCTTCCTGAACTTCGGCTTTG CCAACCGGTTCAGCAAGCCTAAGGGCCCCAGAAATCCTCCTCCTACATGGAACA TC (SEQ ID NO: 24) Zap70 SH2 1, ATGCCTGATCCTGCTGCTCATCTGCCATTCTTCTACGGCAGCATCAGCAGAGCCG SH2 2 domains AGGCCGAAGAACATCTGAAGCTGGCCGGAATGGCCGACGGACTGTTTCTGCTCA GACAGTGCCTGAGAAGCCTCGGCGGCTATGTGCTGTCTCTGGTGCACGATGTGC GGTTCCATCACTTCCCCATCGAGAGACAGCTGAACGGCACCTACGCTATCGCTG GCGGAAAAGCCCATTGTGGACCTGCCGAGCTGTGCGAGTTCTACAGCAGAGATC CTGACGGCCTGCCTTGCAACCTGCGGAAGCCTTGCAATAGACCCAGCGGCCTGG AACCTCAGCCTGGCGTTTTCGACTGCCTGAGAGATGCCATGGTCCGAGATTACG TGCGGCAGACCTGGAAGCTGGAAGGCGAAGCTCTGGAACAGGCAATCATCAGC CAGGCTCCTCAGGTGGAAAAGCTGATCGCCACAACAGCCCACGAGCGGATGCC TTGGTATCACAGCTCCCTGACCAGAGAGGAAGCCGAGCGGAAGCTGTATTCTGG CGCCCAGACCGATGGCAAGTTCCTGCTGAGGCCCAGAAAAGAGCAGGGCACAT ACGCCCTGAGCCTGATCTACGGCAAGACCGTGTACCACTACCTGATCTCCCAGG ACAAGGCCGGCAAGTACTGTATCCCTGAGGGCACCAAGTTCGACACCCTGTGGC AGCTGGTGGAATACCTGAAGCTGAAAGCCGATGGACTGATCTACTGCCTGAAAG AGGCCTGC (SEQ ID NO: 25) Zap70 delta ATGCCTGATCCTGCTGCTCATCTGCCATTCTTCTACGGCAGCATCAGCAGAGCCG kinase AGGCCGAAGAACATCTGAAGCTGGCCGGAATGGCCGACGGACTGTTTCTGCTCA GACAGTGCCTGAGAAGCCTCGGCGGCTATGTGCTGTCTCTGGTGCACGATGTGC GGTTCCATCACTTCCCCATCGAGAGACAGCTGAACGGCACCTACGCTATCGCTG GCGGAAAAGCCCATTGTGGACCTGCCGAGCTGTGCGAGTTCTACAGCAGAGATC CTGACGGCCTGCCTTGCAACCTGCGGAAGCCTTGCAATAGACCCAGCGGCCTGG AACCTCAGCCTGGCGTTTTCGACTGCCTGAGAGATGCCATGGTCCGAGATTACG TGCGGCAGACCTGGAAGCTGGAAGGCGAAGCTCTGGAACAGGCAATCATCAGC CAGGCTCCTCAGGTGGAAAAGCTGATCGCCACAACAGCCCACGAGCGGATGCC TTGGTATCACAGCTCCCTGACCAGAGAGGAAGCCGAGCGGAAGCTGTATTCTGG CGCCCAGACCGATGGCAAGTTCCTGCTGAGGCCCAGAAAAGAGCAGGGCACAT ACGCCCTGAGCCTGATCTACGGCAAGACCGTGTACCACTACCTGATCTCCCAGG ACAAGGCCGGCAAGTACTGTATCCCTGAGGGCACCAAGTTCGACACCCTGTGGC AGCTGGTGGAATACCTGAAGCTGAAAGCCGATGGACTGATCTACTGCCTGAAAG AGGCCTGTCCTAACAGCAGCGCCAGCAATGCTAGCGGAGCTGCTGCACCTACAC TGCCTGCTCACCCTAGCACACTGACACACCCTCAGCGGAGAATCGATACCCTGA ACAGCGACGGCTACACCCCAGAACCTGCCAGAATCACTAGCCCCGACAAGCCC AGACCTATGCCTATGGACACCTCCGTGTACGAGAGCCCCTACAGCGATCCCGAG GAACTGAAGGACAAGAAGCTGTTCCTCAAGCGGGACAACCTG (SEQ ID NO: 26) Zap70 Y492F ATGCCTGATCCTGCTGCTCATCTGCCATTCTTCTACGGCAGCATCAGCAGAGCCG Y493F AGGCCGAAGAACATCTGAAGCTGGCCGGAATGGCCGACGGACTGTTTCTGCTCA GACAGTGCCTGAGAAGCCTCGGCGGCTATGTGCTGTCTCTGGTGCACGATGTGC GGTTCCATCACTTCCCCATCGAGAGACAGCTGAACGGCACCTACGCTATCGCTG GCGGAAAAGCCCATTGTGGACCTGCCGAGCTGTGCGAGTTCTACAGCAGAGATC CTGACGGCCTGCCTTGCAACCTGCGGAAGCCTTGCAATAGACCCAGCGGCCTGG AACCTCAGCCTGGCGTTTTCGACTGCCTGAGAGATGCCATGGTCCGAGATTACG TGCGGCAGACCTGGAAGCTGGAAGGCGAAGCTCTGGAACAGGCAATCATCAGC CAGGCTCCTCAGGTGGAAAAGCTGATCGCCACAACAGCCCACGAGCGGATGCC TTGGTATCACAGCTCCCTGACCAGAGAGGAAGCCGAGCGGAAGCTGTATTCTGG CGCCCAGACCGATGGCAAGTTCCTGCTGAGGCCCAGAAAAGAGCAGGGCACAT ACGCCCTGAGCCTGATCTACGGCAAGACCGTGTACCACTACCTGATCTCCCAGG ACAAGGCCGGCAAGTACTGTATCCCTGAGGGCACCAAGTTCGACACCCTGTGGC AGCTGGTGGAATACCTGAAGCTGAAAGCCGATGGACTGATCTACTGCCTGAAAG AGGCCTGTCCTAACAGCAGCGCCAGCAATGCTAGCGGAGCTGCTGCACCTACAC TGCCTGCTCACCCTAGCACACTGACACACCCTCAGCGGAGAATCGATACCCTGA ACAGCGACGGCTACACCCCAGAACCTGCCAGAATCACTAGCCCCGACAAGCCC AGACCTATGCCTATGGACACCTCCGTGTACGAGAGCCCCTACAGCGATCCCGAG GAACTGAAGGACAAGAAGCTGTTCCTCAAGCGGGACAACCTGCTGATTGCCGA CATCGAACTCGGCTGCGGCAATTTTGGATCTGTGCGGCAGGGCGTGTACCGGAT GCGGAAGAAACAGATCGACGTGGCCATCAAGGTGCTGAAGCAGGGAACCGAGA AGGCCGACACCGAGGAAATGATGCGGGAAGCCCAGATTATGCACCAGCTGGAC AACCCCTACATCGTGCGGCTGATCGGAGTGTGTCAAGCCGAGGCTCTGATGCTG GTCATGGAAATGGCAGGCGGAGGCCCTCTGCACAAGTTTCTCGTTGGCAAGCGG GAAGAGATCCCCGTGTCTAATGTGGCCGAGCTGCTCCACCAAGTGTCTATGGGC ATGAAGTACCTGGAAGAGAAGAACTTCGTGCACCGCGACCTGGCCGCCAGAAA TGTGCTGCTGGTCAACAGACACTACGCCAAGATCAGCGACTTCGGCCTGTCTAA AGCCCTGGGCGCCGACGATAGCTTCTTCACAGCTAGAAGCGCCGGAAAGTGGCC CCTGAAGTGGTACGCCCCTGAGTGCATCAACTTCCGCAAGTTCAGCTCCAGATC CGACGTGTGGTCTTACGGCGTGACCATGTGGGAAGCCCTGAGCTACGGCCAGAA ACCTTACAAGAAGATGAAGGGCCCCGAAGTCATGGCCTTCATCGAACAGGGCA AGAGAATGGAATGCCCTCCTGAGTGCCCTCCAGAGCTGTATGCCCTGATGAGCG ATTGCTGGATCTACAAGTGGGAAGATCGGCCCGACTTCCTGACCGTGGAACAGA GAATGCGGGCCTGCTACTACTCCCTGGCCTCCAAAGTTGAGGGACCTCCTGGCA GCACACAGAAAGCCGAAGCTGCTTGTGCT (SEQ ID NO: 27) SHIP-1 PTP GATATGATCACCATCTTCATCGGCACCTGGAATATGGGCAACGCCCCTCCACCT domain AAGAAGATCACAAGCTGGTTTCTGAGCAAAGGCCAGGGAAAGACCAGGGACGA CAGCGCCGATTACATCCCTCACGATATCTACGTGATCGGGACCCAAGAGGACCC TCTGAGCGAGAAAGAGTGGCTGGAAATTCTGAAGCACTCCCTGCAAGAGATCA CCTCCGTGACCTTCAAGACCGTGGCCATCCACACACTGTGGAACATCCGGATCG TGGTGCTGGCCAAGCCTGAGCACGAGAACAGAATCAGCCACATCTGCACCGAC AACGTGAAAACCGGAATCGCCAACACACTGGGCAACAAAGGCGCCGTGGGAGT GTCCTTCATGTTCAACGGCACCAGCCTGGGCTTCGTGAACAGCCACCTGACATC CGGCTCCGAGAAGAAGCTGCGGCGGAACCAGAACTATATGAACATCCTGCGGT TTCTGGCCCTGGGCGACAAGAAGCTGAGCCCCTTCAACATCACCCACCGGTTCA CCCACCTGTTTTGGTTCGGCGACCTGAACTACAGAGTGGACCTGCCTACCTGGG AAGCCGAGACAATCATCCAGAAGATCAAGCAGCAACAGTACGCCGACCTGCTG TCCCACGATCAGCTGCTGACCGAGAGAAGGGAACAGAAAGTGTTCCTGCACTTC GAGGAAGAGGAAATCACATTCGCCCCTACCTACAGATTCGAGCGGCTGACCCG GGATAAGTACGCCTACACCAAGCAGAAAGCCACCGGCATGAAGTACAACCTGC CTTCTTGGTGCGACCGGGTGCTGTGGAAGTCTTACCCTCTGGTGCACGTCGTGTG CCAGTCTTACGGCAGCACCAGCGACATCATGACCAGCGATCACAGCCCTGTGTT CGCCACATTTGAGGCCGGCGTGACCAGCCAGTTTGTGTCC (SEQ ID NO: 28)
Extracellular Ligand Binding Domain
[0102] As used herein, the term "extracellular ligand binding domain" refers to a domain of a chimeric inhibitory protein of the present disclosure that binds to a specific extracellular ligand. Examples of ligand binding domains are known to those having skill in the art and include, but are not limited to, single-chain variable fragments (scFv), natural receptor/ligand domains, and orthogonal dimerization domains such as leucine zippers that engage with a soluble targeting molecule.
[0103] In some embodiments, the extracellular ligand binding domain comprises an antigen-binding domain. Antigen-binding domains of the present disclosure can include any domain that binds to the antigen including, without limitation, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bispecific antibody, a conjugated antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody (sdAb) such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like.
[0104] In some embodiments, the extracellular ligand binding domain comprises an antibody, or antigen-binding fragment thereof. In some embodiments, the extracellular ligand binding domain comprises a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).
[0105] The term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, the amino acid monomers are linearly linked by peptide linkers, including, but not limited to, comprises any of the amino acid sequences shown in Table 2. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45).
TABLE-US-00004 TABLE 2 Peptide Linkers Linker Amino Acid Sequence SEQ ID NO: (G.sub.2S).sub.1 linker GGS 29 (G.sub.2S).sub.2 linker GGSGGS 30 (G.sub.2S).sub.3 linker GGSGGSGGS 31 (G.sub.2S).sub.4 linker GGSGGSGGSGGS 32 (G.sub.2S).sub.5 linker GGSGGSGGSGGSGGS 33 (G.sub.3S).sub.1 linker GGGS 34 (G.sub.3S).sub.2 linker GGGSGGGS 35 (G.sub.3S).sub.3 linker GGGSGGGSGGGS 36 (G.sub.3S).sub.4 linker GGGSGGGSGGGSGGGS 37 (G.sub.3S).sub.5 linker GGGSGGGSGGGSGGGSGGGS 38 (G.sub.4S).sub.1 linker GGGGS 39 (G.sub.4S).sub.2 linker GGGGSGGGGS 40 (G.sub.4S).sub.3 linker GGGGSGGGGSGGGGS 41 (G.sub.4S).sub.4 linker GGGGSGGGGSGGGGSGGGGS 42 (G.sub.4S).sub.5 linker GGGGSGGGGSGGGGSGGGGSGGGGS 43 Whitlow linker GSTSGSGKPGSGEGSTKG 44 linker 2 EAAAKEAAAKEAAAKEAAAK 45
[0106] "Single-chain Fv" or "sFv" or "scFv" includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen-binding. As described in more detail herein, an scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternatively, the scFv comprises of polypeptide chain where in the C-terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain. In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the scFv peptide linker comprises any of the amino acid sequences shown in Table 2. In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When there are two or more scFv linked together, each scFv can be linked to the next scFv with a peptide linked. In some embodiments, each of the one or more scFvs is separated by a peptide linker.
[0107] The "Fab fragment" (also referred to as fragment antigen-binding) contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen-binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in a single-chain Fab molecule.
[0108] "F(ab')2" fragments contain two Fab' fragments joined, near the hinge region, by disulfide bonds. F(ab')2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab') fragments can be dissociated, for example, by treatment with -mercaptoethanol.
[0109] "Fv" fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.
[0110] The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies. Sdabs are fairly stable and easy to express as fusion partner with the Fc chain of an antibody (Harmsen M M, De Haard H J (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol Biotechnol. 77(1): 13-22).
[0111] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.
[0112] In some embodiments, the extracellular ligand binding domain comprises a domain from a receptor, wherein the receptor is selected from the group consisting of TCR, BCR, a cytokine receptor, RTK receptors, serine/threonine kinase receptors, hormone receptors, immunoglobulin superfamily receptors, and TNFR-superfamily of receptors
[0113] In some embodiments, the extracellular ligand binding domain further comprises a dimerization domain. In some embodiments, the ligand binding domain further comprises a cognate dimerization domain.
[0114] As used herein, the term "ligand" refers to a molecule that binds to a site on a cognate protein (i.e., a cognate protein's ligand binding domain), such as a receptor, thereby producing a cellular response/signal, cell-to-cell recognition, and/or cell-to-cell interaction. A ligand may be, for example, one or more diatomic atom (e.g., NO, CO, etc.), small molecule (e.g., a drug, pharmaceutical, simple sugars, nucleotides, nucleotide derivatives, amino acids, amino acid derivatives, small molecule hormones, small-molecule neurotransmitters, etc.), and/or macromolecule (e.g., lipids, polysaccharides, peptides, soluble proteins, cell surface proteins, cytokines, chemokines, hormones, enzymes, etc.). In some embodiments, the ligand is a naturally-occurring biological ligand (i.e., the ligand arises naturally, such as being natively produced by a cell). In other embodiments, the ligand is a non-naturally-occurring or synthetic ligand (i.e., the ligand is produced synthetically such as by chemical synthesis or is engineered to be different in some aspect than a natural ligand, such engineered for expression in a cell that does not typically express the ligand). In some embodiments, a chimeric inhibitory protein can only be activated through binding of a non-naturally-occurring or synthetic ligand. Examples of synthetic ligands include, but are not limited to, drugs, pharmaceuticals, and engineered macromolecules (e.g., synthetic proteins).
[0115] In some embodiments, the extracellular ligand binding domain of a chimeric receptor binds to a ligand selected from a protein complex, a protein, a peptide, a receptor-binding domain, a nucleic acid, a small molecule, and a chemical agent. In some embodiments, the ligand is a cytokine, chemokine, hormone, or enzyme.
[0116] In some embodiments, the ligand is a cell surface ligand. For example, the ligand of a chimeric inhibitory receptor is present or expressed on a non-target cell surface. Cell surface ligands include, but are not limited to, cell surface markers such as cellular differentiation (CD) markers, receptors, proteins, protein complexes, cell membrane components (e.g., integral membrane proteins, cytoskeletal structures, polysaccharides, lipids, and combinations thereof), and molecules that bind to membrane-associated structures (e.g., soluble antibodies that bind to one or more cell surface ligands). In some embodiments, the cell surface ligand is expressed on a cell that further expresses a cognate ligand of the immune receptor. In some embodiments, the ligand of a chimeric inhibitory receptor is a tumor-associated antigen. In some embodiments, the ligand of a chimeric inhibitory receptor is not expressed on a tumor cell. In some embodiments, the ligand of a chimeric inhibitory receptor is expressed on a non-tumor cell. In some embodiments the ligand of a chimeric inhibitory receptor is expressed on cells of a healthy, or generally considered to be healthy, tissue.
[0117] In an illustrative example, chimeric inhibitory receptors are useful as NOT-logic gates for controlling cell activity, such as immune cell activity. Combinations of activating chimeric receptors and chimeric inhibitory receptors, such as those described herein, can be used in the same cell to reduce on-target off-target toxicity. For instance, if a non-target cell expresses both a ligand that is recognized by an activating chimeric receptor and a ligand that is recognized by a chimeric inhibitory receptor, an engineered cell expressing the activating chimeric receptor may bind to the non-target cell and lead to off-target signaling responses. However, in such a case, the same engineered cell also expresses the chimeric inhibitory receptor that can bind its cognate ligand on the non-target cell and the inhibitory function of the chimeric inhibitory receptor can reduce, decrease, prevent, or inhibit signaling meditated by the activating chimeric receptor ("NOT-logic gating").
[0118] In some embodiments, chimeric inhibitory receptors of the present disclosure specifically bind to one or more ligands that are expressed on normal cells (e.g., cells generally considered healthy) but not on tumor cells. In an illustrative non-limiting examples, combinations of tumor-targeting activating chimeric receptors and chimeric inhibitory receptors can be used in the same immunoresponsive cell to reduce on-target off-tumor toxicity. For instance, if a healthy cell expresses both a tumor-associated antigen that is recognized by the tumor-targeting chimeric receptor and an antigen associated with a healthy cell that is recognized by a chimeric inhibitory receptor, an engineered immunoresponsive cell expressing the tumor-targeting chimeric receptor(s) may bind to the healthy cell and lead to off-tumor cellular responses. In such a case, the same engineered immunoresponsive cell also expresses the inhibitory chimeric antigen that can bind its cognate ligand on the healthy cell and the inhibitory function of the chimeric inhibitory receptor can reduce, decrease, prevent, or inhibit the activation of the immunoresponsive cell meditated by the tumor-targeting chimeric receptor.
[0119] As used herein, the term "immune receptor" refers to a receptor that binds to a ligand and causes an immune system response. Binding to a ligand in general causes activation of the immune receptor. T cell activation is an example of immune receptor activation. Examples of immune receptors are known to those having skill in the art and include, but are not limited to, T cell receptors, pattern recognition receptors (PRRs; such as NOD-like receptors (NLRs) and Toll-like receptors (TLRs)), killer activated receptors (KARs), killer inhibitor receptors (KIRs), complement receptors, Fc receptors, B cell receptors, NK cell receptors, and cytokine receptors.
[0120] Membrane Localization Domain
[0121] The chimeric inhibitory receptors include a membrane localization domain. As used herein, the term "membrane localization domain" refers to a region of a chimeric inhibitory receptor of the present disclosure that localizes the receptor to the cell membrane and includes at least a transmembrane domain. In some embodiments, the membrane localization domain of a chimeric receptor further comprises at least a portion of an extracellular domain. In some embodiments, the membrane localization domain further comprises at least a portion of an intracellular domain. In some embodiments, the membrane localization domain further comprises at least a portion of an extracellular domain and at least a portion of an intracellular domain. In some embodiments, the membrane localization domain includes a portion of an extracellular domain, transmembrane domain, and/or intracellular domain that is sufficient to direct or segregate the chimeric inhibitory receptor to a particular domain of the membrane, such as a lipid raft or a heavy lipid raft. In some embodiments, the extracellular ligand binding domain of a chimeric inhibitory receptor is linked to the membrane localization domain through an extracellular linker region, such as the peptide linkers shown in Table 2.
[0122] In some embodiments, the membrane localization domain comprises a transmembrane domain selected from an LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain from a LAT mutant (see e.g., Pavel Otahal et al., Biochim Biophys Acta. 2011 February; 1813(2):367-76), a BTLA transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIR1 transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAGS transmembrane domain, a LAIR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, an SIRPa transmembrane domain, an HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain.
[0123] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, without limitation, NCBI Reference Nos. NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, without limitation, NCBI Reference Nos. NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, without limitation, NCBI Reference Nos. NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, without limitation, NCBI Reference Nos. NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide may be used. Exemplary 4-1BB polypeptides include, without limitation, NCBI Reference Nos. NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide may be used. Exemplary OX40 polypeptides include, without limitation, NCBI Reference Nos. NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, without limitation, NCBI Reference Nos. NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, without limitation, NCBI Reference Nos. NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, without limitation, NCBI Reference Nos. NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, without limitation, NCBI Reference Nos. NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, without limitation, NCBI Reference Nos. NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Exemplary BTLA polypeptides include, without limitation, NCBI Reference Nos. NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, without limitation, NCBI Reference Nos. NP_001075106.2 and NP_001075107.2.
[0124] In some embodiments, the transmembrane domain comprises a polypeptide comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI Reference No. NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or fragments thereof. In some embodiments, the homology may be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a consecutive portion of NCBI Reference No. NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length.
[0125] Further examples of suitable polypeptides from which a transmembrane domain may be derived include, without limitation, the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R.alpha., ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, and NG2C.
[0126] In some embodiments, the transmembrane domain derived from a LAT mutant is derived from a LAT(CA) mutant. See e.g., Kosugi A., et al. Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 June; 14(6): 669-80.
[0127] In some embodiments, the transmembrane domain is selected from the amino acid sequences shown in Table 3. In some embodiments, the transmembrane domain comprises a polypeptide comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to any of the sequences shown in Table 3. In some embodiments, the homology may be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the transmembrane domain is a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% identical to any of the nucleic acid sequences listed in Table 3. In some embodiments, the transmembrane domain is a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 3.
TABLE-US-00005 TABLE 3 Transmembrane Domains TM domain Amino Acid Sequence DNA Sequence CD28 FWVLVVVGGVLACYSLLVT TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT VAFIIFWV (SEQ ID NO: GCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTT 65) CTGGGTG (SEQ ID NO: 73) PAG GPAGSLLGSGQMQITLWGS GGTCCGGCTGGCTCTCTGCTCGGCAGTGGTCAGATG LAAVAIFFVITFLIFLCSSCD CAGATTACGTTGTGGGGCAGTTTGGCAGCCGTCGCA REKKPR (SEQ ID NO: 66) ATCTTCTTTGTTATCACTTTTCTTATCTTTCTCTGTTC CTCATGTGACAGAGAGAAAAAGCCCCGA (SEQ ID NO: 74) mLAT MEADALSPVGLGLLLLPFL ATGGAAGCCGATGCTCTGTCTCCTGTTGGCCTGGGA VTLLAALCVRCRELPVS CTGCTGCTCCTGCCTTTTCTGGTTACACTGCTGGCCG (SEQ ID NO: 67) CTCTGTGTGTGCGGTGTAGAGAACTGCCAGTTAGT (SEQ ID NO: 75) mLAT(ca) MEADALSPVGLGLLLLPFL ATGGAAGCCGATGCTCTGTCTCCTGTTGGCCTGGGA VTLLAALAVRARELPVS CTGCTGCTCCTGCCTTTTCTCGTTACACTGCTGGCCG (SEQ ID NO: 68) CTCTGGCTGTGCGAGCTAGAGAACTGCCTGTGTCT (SEQ ID NO: 76) LAT (1-33) EEAILVPCVLGLLLLPILAM GAGGAAGCAATCCTGGTGCCGTGTGTACTTGGTCTG LMALCVHCHRLP (SEQ ID CTTTTGTTGCCAATACTTGCGATGCTCATGGCTCTCT NO: 69) GCGTACATTGCCATCGGCTTCCG (SEQ ID NO: 77) LAX IFSGFAGLLAILLVVAVFCIL ATCTTCAGCGGCTTTGCCGGACTGCTGGCTATCCTGC (SEQ ID NO: 70) TGGTTGTGGCCGTGTTCTGTATCCTT (SEQ ID NO: 78) CD3z LCYLLDGILFIYGVILTALFL CTGTGCTACCTGCTGGACGGCATCCTGTTTATCTACG (SEQ ID NO: 71) GCGTGATCCTGACAGCCCTGTTCCTT (SEQ ID NO: 79) CD45 ALIAFLAFLIIVTSIALLVVL GCCCTGATTGCCTTCCTGGCCTTTCTGATCATCGTGA (SEQ ID NO: 72) CCAGCATTGCCCTGCTGGTCGTGCTG (SEQ ID NO: 80)
[0128] In some embodiments, the membrane localization domain further comprises at least a portion of a corresponding extracellular domain and/or at least a portion of a corresponding intracellular domain (see, e.g., spacers and hinges described herein derived from the membrane localization domains described herein).
[0129] In some embodiments, the membrane localization domain further comprises proximal protein fragments. Proximal protein fragments refer to protein segments immediately adjacent to transmembrane domains in their native context. For example, proximal protein fragments can be protein segments that fall outside a transmembrane domain of a protein or that fall outside the conventional boundary of a sequence considered to be a transmembrane domain of a protein. In some embodiments, proximal protein fragments can be a spacer or hinge sequence. In some embodiments, proximal protein fragments can be distinct from a spacer or hinge sequence.
[0130] In some embodiments, the membrane localization domain directs or segregates the chimeric inhibitory receptor to a domain of a cell membrane. As used herein, the term "domain of a cell membrane" refers to a lateral inhomogeneity in lipid composition and physical properties in a cell membrane. Cell membrane domain formation may be driven by multiple forces: hydrogen bonding, hydrophobic entropic forces, charge pairing and van der Waals forces. Cell membrane domains may arise via protein-protein interactions within membranes, protein-lipid interactions within membranes, or lipid-lipid interactions within membranes. Examples of cell membrane domains are known to those having skill in the art and include, but are not limited to, lipid rafts, heavy lipid rafts, light lipid rafts, caveolae, patches, posts, fences, lattices, rafts, and scaffolds. See e.g., Nicolson G. L., The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years, Biochim. Biophys. Acta. 2014 June; 1838(6): 1451-66.
[0131] In some embodiments, the membrane localization domain localizes a chimeric inhibitory receptor of the present disclosure to a lipid raft. In some embodiments, the membrane localization domain interacts with one or more cell membrane components localized in a domain of a cell membrane. Examples of cell membrane components are known to those having skill in the art and include, but are not limited to, various integral membrane proteins, cytoskeletal structures, polysaccharides, lipids, and combinations thereof. See e.g., Nicolson G. L., The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years, Biochim. Biophys. Acta. 2014 June; 1838(6): 1451-66.
[0132] In some embodiments, the membrane localization domain mediates basal localization (i.e., localization in the absence of cognate ligand) of the chimeric inhibitory receptor to a domain of a cell membrane that is distinct from domains of the cell membrane occupied by one or more components of an immune receptor, such as a membrane portion distinct from a lipid raft occupied by an immune receptor. In some embodiments, the basal membrane localization domain is sufficient to mitigate constitutive inhibition of immune receptor activation by the enzymatic inhibitory domain.
[0133] As used herein, the term "immune receptor activation" refers to an event that initiates a signaling cascade that ultimately results in an immune response. T cell activation is an example of immune receptor activation. In general, and without wishing to be bound by theory, while a membrane localization domain can mitigate constitutive inhibition of an immune receptor, binding between the chimeric inhibitory receptor and its cognate ligand generally mediates spatial recruitment of the enzymatic inhibitory domain to be proximal to the immune receptor and/or downstream signaling complexes such that the enzymatic inhibitory domain is capable of negatively regulating an intracellular signal transduction cascade. In a non-limiting illustrative example, binding between the chimeric inhibitory receptor and its cognate ligand can localize the receptor and enzymatic inhibitory domain to an immunological synapse and inhibit immune receptor signaling and/or activation, such as T cell activation (e.g., a inhibit a TCR present in the immunological synapse, such an TCRs bound to its cognate ligand), either directly acting on the immune receptor and/or on another signaling component involved in an intracellular signal transduction cascade.
[0134] In some embodiments, a non-specific transmembrane domain will be sufficient to prevent the enzymatic inhibitory domain from constitutively inhibiting T cell activation. In other embodiments, a transmembrane domain (including proximal protein fragments) can be selected that mediates localization to regions of the cell membrane that are physically distinct from those regions occupied by components of the T-cell receptor (e.g., segregation to "heavy" lipid rafts, instead of "classical" lipid rafts; see e.g., Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 September; 137(1): 1-19), such as regions of the cell membrane other than an immunological synapse.
Spacers and Hinge Domains
[0135] Chimeric inhibitory receptors can also contain spacer or hinge domains. In some embodiments, a spacer domain or a hinge domain is located between an extracellular domain (e.g., comprising the extracellular ligand binding domain) and a transmembrane domain of an chimeric inhibitory receptor, or between an intracellular signaling domain and a transmembrane domain of the chimeric inhibitory receptor. A spacer or hinge domain is any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the intracellular signaling domain in the polypeptide chain. Spacer or hinge domains can provide flexibility to the chimeric inhibitory receptor, or domains thereof, or prevent steric hindrance of the chimeric inhibitory receptor, or domains thereof. In some embodiments, a spacer domain or hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more spacer domain(s) may be included in other regions of an chimeric inhibitory receptor. In some embodiments, a spacer or hinge domain includes at least a portion of an extracellular domain and/or at least a portion of an intracellular domain from the same source as the membrane localization domain.
[0136] Exemplary spacer or hinge domain protein sequences are shown in Table 4. Exemplary spacer or hinge domain nucleotide sequences are shown in Table 5. In some embodiments, a spacer or hinge domain is an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table 4. In some embodiments, a spacer or hinge domain is a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% identical to any of the nucleic acid sequences listed in Table 5. In some embodiments, a spacer or hinge domain is a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 5.
TABLE-US-00006 TABLE 4 Spacer/Hinge Domain Amino Acid Sequences SEQ Amino Acid Sequence ID NO: Description AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP 46 CD28 hinge GPSKP ESKYGPPCPSCP 47 IgG4 minimal hinge ESKYGPPAPSAP 48 IgG4 minimal hinge, no disulfides ESKYGPPCPPCP 49 IgG4 S228P minimal hinge, enhanced disulfide formation EPKSCDKTHTCP 50 IgG1 minimal hinge AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPE 51 Extended CD8a hinge ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL SLVITLYCNHRN TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTR 52 CD8a hinge GLDFACD ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCE 53 LNGFR hinge PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEA DDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFS CQDKQNTVCEECPDGTYSDEADAEC ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC 54 Truncated LNGFR hinge (TNFR- Cys1) AVGQDTQEVIVVPHSLPFKV 55 PDGFR-beta extracellular linker
TABLE-US-00007 TABLE 5 Spacer/Hinge Domain Nucleic Acid Sequences Nucleic Acid Sequence SEQ ID NO: Description GCAGCAGCTATCGAGGTGATGTATCCTCCGCCCTA 56 CD28 hinge CCTGGATAATGAAAAGAGTAATGGGACTATCATTC ATGTAAAAGGGAAGCATCTTTGTCCTTCTCCCCTTT TCCCCGGTCCGTCTAAACCT GAA AGC AAG TAC GGT CCA CCT TGC CCT AGC 57 IgG4 minimal hinge TGT CCG GAA TCC AAG TAC GGC CCC CCA GCG CCT AGT 58 IgG4 minimal hinge, no GCC CCA disulfides GAA TCT AAA TAT GGC CCG CCA TGC CCG CCT 59 IgG4 S228P minimal hinge, TGC CCA enhanced disulfide formation GAA CCG AAG TCT TGT GAT AAA ACT CAT ACG 60 IgG1 minimal hinge TGC CCG GCT GCT GCT TTC GTA CCC GTG TTC CTC CCT 61 Extended CD8a hinge GCT AAG CCT ACG ACT ACC CCC GCA CCG AGA CCA CCC ACG CCA GCA CCC ACG ATTGCT AGC CAG CCC CTT AGT TTG CGA CCA GAA GCT TGT CGG CCT GCT GCT GGT GGC GCG GTA CAT ACC CGC GGC CTT GAT TTT GCTTGC GAT ATA TAT ATC TGG GCG CCT CTG GCC GGA ACA TGC GGG GTC CTC CTC CTT TCT CTG GTT ATT ACT CTC TAC TGT AAT CACAGG AAT GCC TGC CCG ACC GGG CTC TAC ACT CAT AGC 62 LNGFR hinge GGG GAA TGT TGT AAG GCA TGT AAC TTG GGT GAG GGC GTC GCA CAG CCC TGC GGAGCT AAC CAA ACA GTG TGC GAA CCC TGC CTC GAT AGT GTG ACG TTC TCT GAT GTT GTA TCA GCT ACA GAG CCT TGC AAA CCA TGTACT GAG TGC GTT GGA CTT CAG TCA ATG AGC GCT CCA TGT GTG GAG GCA GAT GAT GCG GTC TGT CGA TGT GCT TAC GGA TAC TACCAA GAC GAG ACA ACA GGG CGG TGC GAG GCC TGT AGA GTT TGT GAG GCG GGC TCC GGG CTG GTG TTT TCA TGT CAA GAC AAG CAAAAT ACG GTC TGT GAA GAG TGC CCT GAT GGC ACC TAC TCA GAC GAA GCA GAT GCA GAA TGC GCC TGC CCT ACA GGA CTC TAC ACG CAT AGC 63 Truncated LNGFR hinge (TNFR- GGT GAG TGT TGT AAA GCA TGC AAC CTC GGG Cys1) GAA GGT GTA GCC CAG CCA TGC GGG GCT AAC CAA ACC GTT TGC GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGG 64 PDGFR-beta extracellular TGCCACACTCCTTGCCCTTTAAGGTG linker
[0137] In some embodiments, the chimeric inhibitory receptor further comprises a spacer region between the extracellular ligand binding domain and the membrane localization domain, also referred to as an extracellular linker. In some embodiments, the extracellular linker region is positioned between the extracellular ligand binding domain and membrane localization domain and operably and/or physically linked to each of the extracellular ligand binding domain and the membrane localization domain.
[0138] In some embodiments, the chimeric inhibitory receptor further comprises a spacer region between the membrane localization domain and the enzymatic inhibitory domain, also referred to as an intracellular spacer region. In some embodiments, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the membrane localization domain and the enzymatic inhibitory domain and operably and/or physically linked to each of the membrane localization domain and the enzymatic inhibitory domain.
[0139] In some embodiments, the extracellular linker region and/or intracellular spacer region is derived from a protein selected from the group consisting of: CD8.alpha., CD4, CD7, CD28, IgG1, IgG4, Fc.gamma.RIII.alpha., LNGFR, and PDGFR. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence selected from the group consisting of:
TABLE-US-00008 (SEQ ID NO: 46) AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP, (SEQ ID NO: 47) ESKYGPPCPSCP, (SEQ ID NO: 48) ESKYGPPAPSAP, (SEQ ID NO: 49) ESKYGPPCPPCP, (SEQ ID NO: 50) EPKSCDKTHTCP, (SEQ ID NO: 51) AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN, (SEQ ID NO: 52) TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD. (SEQ ID NO: 53) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSAT EPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEA GSGLVFSCQDKQNTVCEECPDGTYSDEADAEC, (SEQ ID NO: 54) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC, and (SEQ ID NO: 55) AVGQDTQEVIVVPHSLPFKV.
[0140] In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:46. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:47. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:48. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:49. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:50. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:51. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:52. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:53. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:54. In some embodiments, the extracellular linker region and/or intracellular spacer region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:55.
[0141] In some embodiments, the extracellular linker region and/or intracellular spacer region includes a peptide linker, such as any of the amino acid sequences shown in Table 2. In some embodiments, the extracellular linker region and/or intracellular spacer region includes a peptide linker having the amino acid sequence selected from the group consisting of GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45).
[0142] In some embodiments, the extracellular linker region and/or intracellular spacer region modulates sensitivity of the chimeric inhibitory receptor. In some embodiments, the extracellular linker region and/or intracellular spacer region increases sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region reduces sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region modulates potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region increases potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region reduces potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region modulates basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor expressed on the engineered cell relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region reduces basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region. In some embodiments, the extracellular linker region and/or intracellular spacer region increases basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the extracellular linker region and/or intracellular spacer region.
[0143] In some embodiments, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the transmembrane domain and the intracellular signaling domain and is operably linked to each of the transmembrane domain and the intracellular signaling domain. In some embodiments, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the transmembrane domain and the intracellular signaling domain and is physically linked to each of the transmembrane domain and the intracellular signaling domain.
[0144] In some embodiments, the intracellular spacer region modulates sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region increases sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region reduces sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region modulates potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.
[0145] In some embodiments, the intracellular spacer region increases potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region reduces potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region modulates basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor expressed on the engineered cell when expressed on an engineered cell relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region reduces basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region. In some embodiments, the intracellular spacer region increases basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.
Intracellular Inhibitory Co-signaling Domains
[0146] In some embodiments, the chimeric inhibitory receptors comprises one or more intracellular inhibitory co-signaling domains. In some embodiments, the one or more intracellular inhibitory co-signaling domains are between the membrane localization domain and the enzymatic inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains are between the transmembrane domain and the and the enzymatic inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains are C-terminal of the enzymatic inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., a membrane localization, a transmembrane domain, or an enzymatic inhibitory domain) through a peptide linker (e.g., see Table 2) or a spacer or hinge sequence (e.g., see Table 4). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains can be linked through a peptide linker (e.g., see Table 2) or a spacer or hinge sequence (e.g., see Table 4).
[0147] In some embodiments, the one or more intracellular inhibitory co-signaling domains of a chimeric protein comprises one or more ITIM-containing protein, or fragment(s) thereof. ITIMs are conserved amino acid sequences found in cytoplasmic tails of many inhibitory immune receptors. In some embodiments, the one or more ITIM-containing protein, or fragments thereof, is selected from PD-1, CTLA4, TIGIT, BTLA, and LAIR1. In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or a fragment(s) thereof. In some embodiments, the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP, LAGS, HAVR, GITR, and PD-L1. In some embodiments, the inhibitory mechanisms of the enzymatic inhibitory domain and the ITIM and/or non-ITIM scaffolds overlap, e.g., an ITIM-containing protein recruits the endogenous version of the enzyme from which the enzymatic inhibitory domain is derived, such as SHP-1. In some embodiments, the inhibitory mechanisms of the enzymatic inhibitory domain and the ITIM and/or non-ITIM scaffolds are distinct and can be complementary/synergistic, e.g., the activities of an ITIM-containing protein and a Csk or CBL-b derived enzymatic inhibitory domain.
Immune Receptors
[0148] In some embodiments, the immune receptor is a naturally-occurring immune receptor. In some embodiments, the immune receptor is a naturally-occurring antigen receptor. In some embodiments, the immune receptor is selected from a T cell receptor (TCR), a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), an NK cell receptor, a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor. In some embodiments, the immune receptor is a TCR.
In some embodiments, the immune receptor is a chimeric immune receptor. In some embodiments, the immune receptor is a chimeric antigen receptor (CAR). In general, as used herein and unless otherwise specified, immune receptors in a CAR format refer to activating CARs that typically are a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. A CAR of the present disclosure may be a first, second, or third generation CAR. "First generation" CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. "First generation" CARs generally have the intracellular signaling domain from the CD3-zeta (CD3.zeta.) chain, which is the primary transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3.zeta. chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. "Second generation" CARs provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3.zeta.). Preclinical studies have indicated that "Second Generation" CARs can improve the anti-tumor activity of immunoresponsive cell, such as a T cell. "Third generation" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3.zeta.).
[0149] In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is chosen from 4-1BB (i.e., CD 137), CD27, ICOS, and/or CD28. In some embodiments, the CAR. comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence (also referred to as a signal sequence) at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., an scFv) during cellular processing and localization of the CAR to the cellular membrane.
[0150] Various chimeric antigen receptors are known in the art including, but not limited to, ScFv-Fc.epsilon.RI.gamma.CAIX, ScFv-Fc.epsilon.RI.gamma., ScFv-CD3.zeta.; ScFv-CD28-CD3.zeta.; ScFv-CD28-CD3.zeta., ScFv-CD3.zeta.; ScFv-CD4-CD3.zeta.; CD3.zeta./CD137/CD28, ScFv-CD28-41BB-CD3.zeta.; ScFv-CD8-CD3.zeta., ScFv-FceRI.gamma., CD28/4-1BB-CD3.zeta., ScFv-CD28mut-CD3.zeta., Heregulin-CD3.zeta.; ScFv-CD28, ScFv-CD28-OX40-CD3.zeta., ScFv-CD3.zeta., IL-13-CD28-4-1BB-CD3.zeta., IL-13-CD3; IL-13-CD3; ScFv-Fc.epsilon.RI.gamma., ScFV-CD4-Fc.epsilon.RI.gamma., ScFV-CD28-Fc.epsilon.RI.gamma., Ly49H-CD3.zeta., NKG2D-CD3.zeta., ScFv-b2c-CD3.zeta., and FceRI-CD28-CD3.zeta.. In some embodiments, the chimeric antigen receptor has been modified to include control elements. In some embodiments, the chimeric antigen receptor is a split chimeric antigen receptor; see e.g., WO2017/091546.
[0151] In some embodiments, the immune receptor is a chimeric TCR. A chimeric TCR generally includes an extracellular ligand binding domain grafted onto one or more constant domains of a TCR chain, for example a TCR alpha chain or TCR beta chain, to create a chimeric TCR that binds specifically to an antigen of interest, such a tumor-associated antigen. Without wishing to be bound by theory, it is believed that chimeric TCRs may signal through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., scFv) can be grafted to the constant domain (e.g., at least a portion of the extracellular constant domain, the transmembrane domain and cytoplasmic domain) of a TCR chain, such as the TCR alpha chain and/or the TCR beta chain. As another example, the CDRs of an antibody or antibody fragment may be grafted into a TCR alpha chain and/or beta chain to create a chimeric TCR that binds specifically to an antigen. Such chimeric TCRs may be produced by methods known in the art (e.g., Willemsen R A et al., Gene Therapy 2000; 7:1369-1377; Zhang T et al., Cancer Gene Ther 2004 11: 487-496; and Aggen et al., Gene Ther. 2012 April; 19(4): 365-74; herein incorporated by reference for all purposes).
[0152] The antigen of an immune receptor, such as a chimeric antigen receptor, can be a tumor-associated antigen.
[0153] Immune receptors generally are capable of inducing signal transduction or changes in protein expression in the immune receptor-expressing cell that results in the modulation of an immune response upon binding to a cognate ligand (e.g., regulate, activate, initiate, stimulate, increase, prevent, attenuate, inhibit, reduce, decrease, inhibit, or suppress an immune response). For example, when CD3 chains present in a TCR/CAR cluster in response to ligand binding, an immunoreceptor tyrosine-based activation motifs (ITAMs)-meditated signal transduction cascade is produced. Specifically, in certain embodiments, when an endogenous TCR, exogenous TCR, chimeric TCR, or a CAR (specifically an activating CAR) binds their respective antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g. CD4 or CD8, CD3.gamma./.delta./.epsilon./.zeta., etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated that in turn can initiate a T cell activation pathway and ultimately activates transcription factors, such as NF-.kappa.B and AP-1. These transcription factors are capable of inducing global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response, such as cytokine production and/or T cell mediated killing.
Nucleic Acids Encoding Chimeric Inhibitory Receptors
[0154] Provided herein, in other aspects, are nucleic acids encoding at least one chimeric inhibitory receptor as described above. In some embodiments, the nucleic acid encoding the at least one chimeric inhibitory receptor is a vector. In some embodiments, the vector is selected from a plasmid vector, a viral vector, a lentiviral vector, or a phage vector.
[0155] When the chimeric inhibitory receptor is a multichain receptor, a set of polynucleotides is used. In this case, the set of polynucleotides can be cloned into a single vector or a plurality of vectors. In some embodiments, the polynucleotide comprises a sequence encoding a chimeric inhibitory receptor, wherein the sequence encoding an extracellular ligand binding domain is contiguous with and in the same reading frame as a sequence encoding an intracellular signaling domain and a membrane localization domain.
[0156] The polynucleotide can be codon optimized for expression in a mammalian cell. In some embodiments, the entire sequence of the polynucleotide has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148, herein incorporated by reference for all purposes.
[0157] The polynucleotide encoding a chimeric inhibitory receptor can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the polynucleotide, by deriving it from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the polynucleotide can be produced synthetically, rather than cloned.
[0158] The polynucleotide can be cloned into a vector. In some embodiments, an expression vector known in the art is used. Accordingly, the present disclosure includes retroviral and lentiviral vector constructs expressing a chimeric inhibitory receptor that can be directly transduced into a cell.
[0159] The present disclosure also includes an RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence ("UTR") (e.g., a 3' and/or 5' UTR described herein), a 5' cap (e.g., a 5' cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail. RNA so produced can efficiently transfect different kinds of cells. In some embodiments, an RNA chimeric inhibitory receptor vector is transduced into a cell, e.g., a T cell or a NK cell, by electroporation.
[0160] In some embodiments, a vector of the present disclosure may further comprise a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator, an element allowing episomal replication, and/or elements allowing for selection.
Engineered Cells
[0161] Also provided herein are genetically engineered cells comprising a nucleic acid encoding at least one chimeric inhibitory receptor of the present disclosure or that express a chimeric inhibitory receptor of the present disclosure. Various ways of introducing nucleic acids/vectors (i.e., genetically engineering) are known to those having skill in the art and include, but are not limited to, transduction (i.e., viral infection), transformation, and transfection. Mechanisms of transfection include chemical-based transfection (e.g., calcium phosphate-mediated, lipofection/liposome mediated, etc.), non-chemical-based transfection (e.g., electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, etc.), and particle-based transfection (e.g., gene gun, magnetofection, particle bombardment, etc.).
[0162] In some embodiments, a genetically engineered cell of the present disclosure is an immunomodulatory cell. Immunomodulatory cells include, but are not limited to, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.
[0163] In some embodiments, a genetically engineered cell of the present disclosure is an immune cell. In some embodiments, the immune cell is a T cell. Examples of T cells include, but are not limited to CD8+ T cells, CD4+ T cells, effector cells, helper cells (T.sub.H cells), cytotoxic cells (T.sub.C cells, CTLs, T-killer cells, killer T cells), memory cells (central memory T cells, effector memory T cells, tissue resident memory T cells, virtual memory T cells, etc.), regulatory T cells (e.g., CD4+, FOXP3+, CD25+), natural killer T cells, mucosal associated invariant cells, and gamma delta T cells. In some embodiments, the immune cell is a
[0164] In some embodiments, a genetically engineered cell of the present disclosure is a stem cell, such as a mesenchymal stem cell (MSC), pluripotent stem cell, embryonic stem cell, adult stem cell, bone-marrow stem cell, umbilical cord stem cells, or other stem cell.
[0165] In some embodiments, a genetically engineered cell is autologous. In some embodiments, a genetically engineered cell is allogeneic.
[0166] In some embodiments, the genetically engineered cell further comprises an immune receptor. In some embodiments, the immune receptor is a naturally-occurring immune receptor (e.g., the genetically engineered is an immune cell expressing an endogenous immune receptor). In some embodiments, the immune receptor is a naturally-occurring antigen receptor. In some embodiments, the immune receptor is selected from a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
[0167] In some embodiments, the immune receptor of the cell is a chimeric immune receptor. In some embodiments, the immune receptor is a chimeric antigen receptor. In some embodiments, the chimeric receptor inhibits immune receptor activation upon ligand binding.
[0168] In some embodiments, the genetically engineered cell is further engineered to express an exogenous immune receptor. For example, the genetically engineered cell can be engineered to express a chimeric immune receptor, such as a CAR. In another example, the genetically engineered cell can be engineered to express a naturally-occurring immune receptor exogenous to the engineered cell.
[0169] In some embodiments, the genetically engineered cell is engineered to express a chimeric inhibitory receptor and an exogenous immune receptor. The genetically engineered cell can be engineered to express both a chimeric inhibitory receptor and an exogenous immune receptor simultaneously (e.g., polynucleotides encoding each receptor are introduced simultaneously). The genetically engineered cell can be engineered to express both a chimeric inhibitory receptor and an exogenous immune receptor sequentially (e.g., first engineered to express either the chimeric inhibitory receptor and the exogenous immune receptor, then subsequently engineered to express the other receptor).
[0170] In some embodiments, ligand binding to a chimeric inhibitory receptor of the present disclosure and cognate immune receptor ligand binding to the immune receptor localizes the chimeric inhibitory receptor proximal to the immune receptor. In some embodiments, localization of the chimeric inhibitory receptor proximal to the immune receptor inhibits immune receptor activation. In some embodiments, immune receptor activation is T cell activation. For example, in the case of T cell signaling and/or activation, respective ligands binding to the chimeric inhibitory receptor and the immune receptor localizes the chimeric inhibitory receptor proximal to the immune receptor in an immunological synapse.
Method of Production and Use
[0171] In another aspect, the present disclosure provides a method of preparing a genetically engineered cell (e.g., a genetically engineered immunomodulatory cell) expressing or capable of expressing a chimeric inhibitory receptor for experimental or therapeutic use. In another aspect, the present disclosure provides a method of preparing a genetically engineered cell (e.g., a genetically engineered immunomodulatory cell) expressing or capable of expressing a chimeric inhibitory receptor and an immune receptor for experimental or therapeutic use.
[0172] Ex vivo procedures for making therapeutic chimeric inhibitory receptor-engineered cells are well known in the art. For example, cells are isolated from a mammal (e.g., a human) and genetically engineered (i.e., transduced or transfected in vitro) with a vector expressing a chimeric inhibitory receptor disclosed herein. The chimeric inhibitory receptor-engineered cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the chimeric inhibitory receptor-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient. The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art, therefore the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of immune effector cells (e.g., T cells, NK cells) comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
[0173] In some embodiments, the methods comprise culturing the population of cells (e.g. in cell culture media) to a desired cell density (e.g., a cell density sufficient for a particular cell-based therapy). In some embodiments, the population of cells are cultured in the absence of an agent that represses activity of the repressible protease or in the presence of an agent that represses activity of the repressible protease.
[0174] In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 2-fold the number of cells of the starting population. In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 4-fold the number of cells of the starting population. In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 16-fold the number of cells of the starting population.
[0175] Also provided herein are methods of inhibiting immune receptor activation. In some embodiments, the method including: contacting a genetically engineered cell comprising a nucleic acid encoding at least one chimeric receptor of the present disclosure, a genetically engineered cell that express a chimeric inhibitory receptor of the present disclosure, or a pharmaceutical composition including the genetically engineered cell with a cognate ligand under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein, when localized proximal to an immune receptor expressed on a cell membrane of the engineered cell, the chimeric inhibitory inhibits immune receptor activation.
[0176] Also provided herein are method for reducing an immune response. In some embodiments, the method comprises: administering a genetically engineered cell comprising a nucleic acid encoding at least one chimeric receptor of the present disclosure, a genetically engineered cell that express a chimeric inhibitory receptor of the present disclosure, or a pharmaceutical composition including the genetically engineered cells to a subject in need of such treatment.
[0177] Also provided herein are method of preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, the method including: administering a genetically engineered immunomodulatory cell comprising a nucleic acid encoding at least one chimeric receptor of the present disclosure, a genetically engineered immunomodulatory cell that express a chimeric inhibitory receptor of the present disclosure, or a pharmaceutical composition including the genetically engineered immunomodulatory cell to a subject in need of such treatment.
[0178] Also provided herein are method of preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, the method including: contacting a genetically engineered immunomodulatory cell comprising a nucleic acid encoding at least one chimeric receptor of the present disclosure, a genetically engineered immunomodulatory cell that express a chimeric inhibitory receptor of the present disclosure, or a pharmaceutical composition including the genetically engineered immunomodulatory cell with a cognate ligand under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand, wherein, when localized proximal to an immune receptor expressed on a cell membrane of the engineered cell, the chimeric inhibitory inhibits immune receptor activation.
[0179] Also provided herein are methods of treating an autoimmune disease or disease treatable by reducing an immune response. In some embodiments, the method includes: administering a genetically engineered cell comprising a nucleic acid encoding at least one chimeric receptor of the present disclosure, genetically engineered cells of the present disclosure that express a chimeric inhibitory receptor, or a pharmaceutical composition including the genetically engineered cell to a subject in need of such treatment.
[0180] In some embodiments, the methods include administering or contacting genetically engineered cells that further express or are capable of expressing an immune receptor. In some embodiments, the methods include administering or contacting genetically engineered cells that are further engineered to express an immune receptor. In some embodiments, the methods include administering or contacting genetically engineered cells that further express or are capable of expressing a chimeric immune receptor. In some embodiments, the methods include administering or contacting genetically engineered cells that are further engineered to express a chimeric immune receptor. In some embodiments, the methods include administering or contacting genetically engineered cells that further express or are capable of expressing a CAR. In some embodiments, the methods include administering or contacting genetically engineered cells that are further engineered to express a CAR.
[0181] Attenuation of an immune response initiated by an immune receptor (e.g., a tumor targeting chimeric receptor) can be a decrease or reduction in the activation of the immune receptor, a decrease or reduction in the signal transduction of the immune receptor, or a decrease or reduction in the activation of the engineered cell. The inhibitory chimeric receptor can attenuate activation of the immune receptor, signal transduction by the immune receptor, or activation of the engineered cell by the immune receptor 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to the activation of the immune receptor, signal transduction, or activation of the engineered cell as compared to an engineered cell lacking an inhibitory chimeric receptor. In some embodiments, attenuation refers to a decrease or reduction of the activity of the immune receptor after it has been activated.
[0182] Prevention of an immune response initiated by an immune receptor (e.g., a tumor targeting chimeric receptor) can be an inhibition or reduction in the activation of the immune receptor, an inhibition or reduction in the signal transduction of the immune receptor, or an inhibition or reduction in the activation of the engineered cell. The inhibitory chimeric receptor can prevent activation of the immune receptor, signal transduction by the immune receptor, or activation of the engineered cell by the immune receptor by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to the activation of the immune receptor, signal transduction, or activation of the engineered cell as compared to an engineered cell lacking an inhibitory chimeric receptor. In some embodiments, prevention refers to a blockage of the activity of the immune receptor before it has been activated.
[0183] Inhibition of an immune response initiated by an immune receptor (e.g., a tumor targeting chimeric receptor) can be an inhibition or reduction in the activation of the immune receptor, an inhibition or reduction in the signal transduction of the immune receptor, or an inhibition or reduction in the activation of the engineered cell. The inhibitory chimeric receptor can inhibit activation of the immune receptor, signal transduction by the immune receptor, or activation of the engineered cell by the immune receptor by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to the activation of the immune receptor, signal transduction, or activation of the engineered cell as compared to an engineered cell lacking an inhibitory chimeric receptor. In some embodiments, inhibition refers to a decrease or reduction of the activity of the immune receptor before or after it has been activated.
[0184] Suppression of an immune response initiated by an immune receptor (e.g., a tumor targeting chimeric receptor) can be an inhibition or reduction in the activation of the immune receptor, an inhibition or reduction in the signal transduction of the immune receptor, or an inhibition or reduction in the activation of the engineered cell. The inhibitory chimeric receptor can suppress activation of the immune receptor, signal transduction by the immune receptor, or activation of the engineered cell by the immune receptor by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more as compared to the activation of the immune receptor, signal transduction, or activation of the engineered cell as compared to an engineered cell lacking an inhibitory chimeric receptor. In some embodiments, suppression refers to a decrease or reduction of the activity of the immune receptor before or after it has been activated.
[0185] The immune response can be cytokine or chemokine production and secretion from an activated immunomodulatory cell. The immune response can be a cell-mediated immune response to a target cell, such as cell-mediated killing.
[0186] In some embodiments, the chimeric inhibitory receptor is capable of suppressing cytokine production from an activated engineered cell, such as an immunomodulatory cell. In some embodiments, the chimeric inhibitory receptor is capable of suppressing a cell-mediated immune response to a target cell, wherein the immune response is induced by activation of the engineered cell.
[0187] In one aspect, the present disclosure provides a type of cell therapy where cells, such as immune cells, are genetically engineered to express a chimeric inhibitory receptor provided herein and the genetically engineered cells are administered to a subject in need thereof.
[0188] Thus, in some embodiments, the methods comprise delivering cells of the expanded population of cells to a subject in need of a cell-based therapy to treat a condition or disorder. In some embodiments, the subject is a human subject. In some embodiments, the condition or disorder is an autoimmune condition. In some embodiments, the condition or disorder is an immune related condition. In some embodiments, the condition or disorder is a cancer (e.g., a primary cancer or a metastatic cancer). In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer.
Pharmaceutical Compositions
[0189] The chimeric inhibitory receptor or genetically engineered cell can be formulated in pharmaceutical compositions. Pharmaceutical compositions of the present disclosure can comprise a chimeric inhibitory receptor (e.g., an iCAR) or genetically engineered cell (e.g., a plurality of chimeric inhibitory receptor-expressing cells), as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. In certain embodiments, the composition is directly injected into an organ of interest (e.g., an organ affected by a disorder). Alternatively, the composition may be provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during, or after administration of the composition to increase production of T cells, NK cells, or CTL cells in vitro or in vivo.
[0190] In certain embodiments, the compositions are pharmaceutical compositions comprising genetically engineered cells, such as immunomodulatory or immune cells, or their progenitors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunomodulatory or immune cells, or progenitors, can be obtained from one subject, and administered to the same subject or a different, compatible subject. In some embodiments, genetically engineered cells, such as immunomodulatory or immune cells, or their progeny may be derived from peripheral blood cells (e.g., in vivo, ex vivo, or in vitro derived) and may be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition containing a genetically engineered cell of the present disclosure), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
[0191] Certain aspects of the present disclosure relate to formulations of compositions comprising chimeric receptors of the present disclosure or genetically engineered cells (e.g., immunomodulatory or immune cells of the present disclosure) expressing such chimeric receptors. In some embodiments, compositions of the present disclosure comprising genetically engineered cells may be provided as sterile liquid preparations, including without limitation isotonic aqueous solutions, suspensions, emulsions, dispersions, and viscous compositions, which may be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions may be more convenient to administer, especially by injection. In some embodiments, viscous compositions can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.
[0192] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
[0193] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required. In some embodiments, compositions of the present disclosure can be isotonic, i.e., having the same osmotic pressure as blood and lacrimal fluid. In some embodiments, the desired isotonicity may be achieved using, for example, sodium chloride, dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.
[0194] In some embodiments, compositions of the present disclosure may further include various additives that may enhance the stability and sterility of the compositions. Examples of such additives include, without limitation, antimicrobial preservatives, antioxidants, chelating agents, and buffers. In some embodiments, microbial contamination may be prevented by the inclusions of any of various antibacterial and antifungal agents, including without limitation parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of an injectable pharmaceutical formulation of the present disclosure can be brought about by the use of suitable agents that delay absorption, such as aluminum monostearate and gelatin. In some embodiments, sterile injectable solutions can be prepared by incorporating genetically modified cells of the present disclosure in a sufficient amount of the appropriate solvent with various amounts of any other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. In some embodiments, the compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing agents, pH buffering agents, and antimicrobials depending upon the route of administration and the preparation desired.
[0195] In some embodiments, the components of the formulations of the present disclosure are selected to be chemically inert and to not affect the viability or efficacy of the genetically modified cells of the present disclosure.
[0196] One consideration concerning the therapeutic use of the genetically engineered cells of the present disclosure is the quantity of cells needed to achieve optimal efficacy. In some embodiments, the quantity of cells to be administered will vary for the subject being treated. In certain embodiments, the quantity of genetically engineered cells that are administered to a subject in need thereof may range from 1.times.10.sup.4 cells to 1.times.10.sup.10 cells. In some embodiments, the precise quantity of cells that would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art based on the present disclosure and the knowledge in the art.
[0197] Whether it is a polypeptide, antibody, nucleic acid, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "therapeutically effective amount" or "prophylactically effective amount" (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
[0198] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Kits
[0199] Certain aspects of the present disclosure relate to kits for the treatment and/or prevention of a cancer or other diseases (e.g., immune-related or autoimmune disorders). In certain embodiments, the kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more chimeric receptors of the present disclosure, isolated nucleic acids of the present disclosure, vectors of the present disclosure, and/or cells of the present disclosure (e.g., genetically engineered cells, such as immunomodulatory or immune cells). In some embodiments, the kit comprises a sterile container. In some embodiments, such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
[0200] In some embodiments, therapeutic or prophylactic composition is provided together with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing a cancer or immune-related disorder. In some embodiments, the instructions may include information about the use of the composition for the treatment and/or prevention of the disorder. In some embodiments, the instructions include, without limitation, a description of the therapeutic or prophylactic composition, a dosage schedule, an administration schedule for treatment or prevention of the disorder or a symptom thereof, precautions, warnings, indications, counter-indications, over-dosage information, adverse reactions, animal pharmacology, clinical studies, and/or references. In some embodiments, the instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
Additional Embodiments
[0201] Provided below are enumerated embodiments describing specific non-limiting embodiments of the present invention:
Embodiment 1. A chimeric inhibitory receptor comprising:
[0202] an extracellular ligand binding domain;
[0203] a membrane localization domain, wherein the membrane localization domain comprises a transmembrane domain; and
[0204] an enzymatic inhibitory domain, wherein the enzymatic inhibitory domain inhibits immune receptor activation when proximal to an immune receptor.
[0205] Embodiment 2. The chimeric inhibitory receptor of embodiment 1, wherein the extracellular ligand binding domain binds to a ligand selected from the group consisting of: a protein complex, a protein, a peptide, a receptor-binding domain, a nucleic acid, a small molecule, and a chemical agent.
[0206] Embodiment 3. The chimeric inhibitory receptor of embodiment 1 or embodiment 2, wherein the extracellular ligand binding domain comprises an antibody, or antigen-binding fragment thereof.
[0207] Embodiment 4. The chimeric inhibitory receptor of embodiment 1 or embodiment 2, wherein the extracellular ligand binding domain comprises a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).
[0208] Embodiment 5. The chimeric inhibitory receptor of any one of embodiments 1-4, wherein the ligand is a tumor-associated antigen.
[0209] Embodiment 6. The chimeric inhibitory receptor of any one of embodiments 1-4, wherein the ligand is not expressed on a tumor cell.
[0210] Embodiment 7. The chimeric inhibitory receptor of any one of embodiments 1-4, wherein the ligand is expressed on a non-tumor cell.
[0211] Embodiment 8. The chimeric inhibitory receptor of any one of embodiments 1-4, wherein the ligand is expressed on cells of a healthy tissue.
[0212] Embodiment 9. The chimeric inhibitory receptor of any one of embodiments 1-8, wherein the extracellular ligand binding domain comprises a dimerization domain.
[0213] Embodiment 10. The chimeric inhibitory receptor of embodiment 9, wherein the ligand further comprises a cognate dimerization domain.
[0214] Embodiment 11. The chimeric inhibitory receptor of any one of embodiments 2-10, wherein the ligand is a cell surface ligand.
[0215] Embodiment 12. The chimeric inhibitory receptor of embodiment 11, wherein the cell surface ligand is expressed on a cell that further expresses a cognate ligand of the immune receptor.
[0216] Embodiment 13. The chimeric inhibitory receptor of any one of embodiments 1-12, wherein the membrane localization domain further comprises at least a portion of an extracellular domain.
[0217] Embodiment 14. The chimeric inhibitory receptor of any one of embodiments 1-12, wherein the membrane localization domain further comprises at least a portion of an intracellular domain.
[0218] Embodiment 15. The chimeric inhibitory receptor of any one of embodiments 1-12, wherein the membrane localization domain further comprises at least a portion of an extracellular domain and at least a portion of an intracellular domain.
[0219] Embodiment 16. The chimeric inhibitory receptor of any one of embodiments 1-12, wherein the membrane localization domain comprises a transmembrane domain selected from the group consisting of: a LAX transmembrane domain, a CD25 transmembrane domain, a CD7 transmembrane domain, a LAT transmembrane domain, a transmembrane domain from a LAT mutant, a BTLA transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3zeta transmembrane domain, a CD4 transmembrane domain, a 4-IBB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a 2B4 transmembrane domain, a PD-1 transmembrane domain, a CTLA4 transmembrane domain, a BTLA transmembrane domain, a TIM3 transmembrane domain, a LIR1 transmembrane domain, an NKG2A transmembrane domain, a TIGIT transmembrane domain, and a LAGS transmembrane domain, a LAIR1 transmembrane domain, a GRB-2 transmembrane domain, a Dok-1 transmembrane domain, a Dok-2 transmembrane domain, a SLAP1 transmembrane domain, a SLAP2 transmembrane domain, a CD200R transmembrane domain, an SIRPa transmembrane domain, an HAVR transmembrane domain, a GITR transmembrane domain, a PD-L1 transmembrane domain, a KIR2DL1 transmembrane domain, a KIR2DL2 transmembrane domain, a KIR2DL3 transmembrane domain, a KIR3DL1 transmembrane domain, a KIR3DL2 transmembrane domain, a CD94 transmembrane domain, a KLRG-1 transmembrane domain, a PAG transmembrane domain, a CD45 transmembrane domain, and a CEACAM1 transmembrane domain.
[0220] Embodiment 17. The chimeric inhibitory receptor of embodiment 16, wherein the membrane localization domain further comprises at least a portion of a corresponding extracellular domain and/or at least a portion of a corresponding intracellular domain.
[0221] Embodiment 18. The chimeric inhibitory receptor of embodiment 16 or embodiment 17, wherein the LAT mutant is a LAT(CA) mutant.
[0222] Embodiment 19. The chimeric inhibitory receptor of any one of embodiments 1-18, wherein the membrane localization domain directs or segregates the chimeric inhibitory receptor to a domain of a cell membrane.
[0223] Embodiment 20. The chimeric inhibitory receptor of any one of embodiments 1-19, wherein the membrane localization domain localizes the chimeric inhibitory receptor to a lipid raft or a heavy lipid raft.
[0224] Embodiment 21. The chimeric inhibitory receptor of any one of embodiments 1-20, wherein the membrane localization domain interacts with one or more cell membrane components localized in a domain of a cell membrane.
[0225] Embodiment 22. The chimeric inhibitory receptor of any one of embodiments 1-21, wherein the membrane localization domain is sufficient to mitigate constitutive inhibition of immune receptor activation by the enzymatic inhibitory domain in the absence of the extracellular ligand binding domain binding a cognate ligand.
[0226] Embodiment 23. The chimeric inhibitory receptor of any one of embodiments 1-21, wherein the membrane localization domain mediates localization of the chimeric inhibitory receptor to a domain of a cell membrane that is distinct from domains of the cell membrane occupied by one or more components of an immune receptor in the absence of the extracellular ligand binding domain binding a cognate ligand.
[0227] Embodiment 24. The chimeric inhibitory receptor of embodiment 23, wherein the membrane localization domain further comprises proximal protein fragments.
[0228] Embodiment 25. The chimeric inhibitory receptor of any one of embodiments 1-24, wherein the chimeric inhibitory receptor further comprises one or more intracellular inhibitory co-signaling domains.
[0229] Embodiment 26. The chimeric inhibitory receptor of embodiment 25, wherein the one or more intracellular inhibitory co-signaling domains comprise one or more ITIM-containing proteins, or fragments thereof.
[0230] Embodiment 27. The chimeric inhibitory receptor of embodiment 26, wherein the one or more ITIM-containing proteins, or fragments thereof, are selected from the group consisting of: PD-1, CTLA4, TIGIT, BTLA, and LAIR1.
[0231] Embodiment 28. The chimeric inhibitory receptor of embodiment 25, wherein the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or fragments thereof.
[0232] Embodiment 29. The chimeric inhibitory receptor of embodiment 28, wherein the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from the group consisting of: GRB-2, Dok-1, Dok-2, SLAP1, SLAP2, LAGS, HAVR, GITR, and PD-L1.
[0233] Embodiment 30. The chimeric inhibitory receptor of any one of embodiments 1-29, wherein the extracellular ligand binding domain is linked to the membrane localization domain through an extracellular linker region.
[0234] Embodiment 31. The chimeric inhibitory receptor of embodiment 30, wherein the extracellular linker region is positioned between the extracellular ligand binding domain and membrane localization domain and operably and/or physically linked to each of the extracellular ligand binding domain and the membrane localization domain.
[0235] Embodiment 32. The chimeric inhibitory receptor of embodiment 30 or embodiment 31, wherein the extracellular linker region is derived from a protein selected from the group consisting of: CD8alpha, CD4, CD7, CD28, IgG1, IgG4, FcgammaRIIIalpha, LNGFR, and PDGFR.
[0236] Embodiment 33. The chimeric inhibitory receptor of embodiment 30 or embodiment 31, wherein the extracellular linker region comprises an amino acid sequence selected from the group consisting of: AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:46), ESKYGPPCPSCP (SEQ ID NO:47), ESKYGPPAPSAP (SEQ ID NO:48), ESKYGPPCPPCP (SEQ ID NO:49), EPKSCDKTHTCP (SEQ ID NO:50), AAAFVPVFLPAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:51), TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:52), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCT ECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ NTVCEECPDGTYSDEADAEC (SEQ ID NO:53), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC (SEQ ID NO:54), and AVGQDTQEVIVVPHSLPFKV (SEQ ID NO:55).
[0237] Embodiment 34. The chimeric inhibitory receptor of embodiment 30 or embodiment 31, wherein the extracellular linker region comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45).
[0238] Embodiment 35. The chimeric inhibitory receptor of any one of embodiments 1-33, wherein the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the membrane localization domain and the enzymatic inhibitory domain and operably and/or physically linked to each of the membrane localization domain and the enzymatic inhibitory domain.
[0239] Embodiment 36. The chimeric inhibitory receptor of embodiment 34, wherein the intracellular spacer region comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 29), GGSGGS (SEQ ID NO: 30), GGSGGSGGS (SEQ ID NO: 31), GGSGGSGGSGGS (SEQ ID NO: 32), GGSGGSGGSGGSGGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGSGGGS (SEQ ID NO: 35), GGGSGGGSGGGS (SEQ ID NO: 36), GGGSGGGSGGGSGGGS (SEQ ID NO: 37), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 44), and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 45).
[0240] Embodiment 37. The chimeric inhibitory receptor of embodiment 34, wherein the intracellular spacer region comprises an amino acid sequence selected from the group consisting of: AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:46), ESKYGPPCPSCP (SEQ ID NO:47), ESKYGPPAPSAP (SEQ ID NO:48), ESKYGPPCPPCP (SEQ ID NO:49), EPKSCDKTHTCP (SEQ ID NO:50), AAAFVPVFLPAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:51), TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:52), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCT ECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ NTVCEECPDGTYSDEADAEC (SEQ ID NO:53), ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC (SEQ ID NO:54), and AVGQDTQEVIVVPHSLPFKV (SEQ ID NO:55).
[0241] Embodiment 38. The chimeric inhibitory receptor of any one of embodiments 1-34, wherein the enzymatic inhibitory domain comprises at least a portion of an extracellular domain, a transmembrane domain, and/or an intracellular domain.
[0242] Embodiment 39. The chimeric inhibitory receptor of embodiment 38, wherein the enzymatic inhibitory domain comprises an enzyme catalytic domain.
[0243] Embodiment 40. The chimeric inhibitory receptor of any one of embodiments 1-34, wherein the enzymatic inhibitory domain comprises at least a portion of an enzyme.
[0244] Embodiment 41. The chimeric inhibitory receptor of embodiment 40, wherein the portion of the enzyme comprises an enzyme domain or an enzyme fragment.
[0245] Embodiment 42. The chimeric inhibitory receptor of embodiment 40, wherein the portion of the enzyme is a catalytic domain of the enzyme.
[0246] Embodiment 43. The chimeric inhibitory receptor of any one of embodiments 39-42, wherein the enzyme is selected from the group consisting of: CSK, SHP-1, SHP-2, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, ZAP70, and RasGAP.
[0247] Embodiment 44. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from CSK.
[0248] Embodiment 45. The chimeric inhibitory receptor of embodiment 44, wherein the enzymatic inhibitory domain comprises a CSK protein with a SRC homology 3 (SH3) deletion.
[0249] Embodiment 46. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from SHP-1.
[0250] Embodiment 47. The chimeric inhibitory receptor of embodiment 47, wherein the enzymatic inhibitory domain comprises a protein tyrosine phosphatase (PTP) domain.
[0251] Embodiment 48. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from SHP-2.
[0252] Embodiment 49. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from PTEN.
[0253] Embodiment 50. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from CD45.
[0254] Embodiment 51. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from CD148.
[0255] Embodiment 52. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from PTP-MEG1.
[0256] Embodiment 53. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from PTP-PEST.
[0257] Embodiment 54. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from c-CBL.
[0258] Embodiment 55. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from CBL-b.
[0259] Embodiment 56. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from PTPN22.
[0260] Embodiment 57. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from LAR.
[0261] Embodiment 58. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from PTPH1.
[0262] Embodiment 59. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from SHIP-1.
[0263] Embodiment 60. The chimeric inhibitory receptor of embodiment 60, wherein the enzymatic inhibitory domain comprises a protein tyrosine phosphatase (PTP) domain.
[0264] Embodiment 61. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from ZAP70.
[0265] Embodiment 62. The chimeric inhibitory receptor of embodiment 58, wherein the enzymatic inhibitory domain comprises a SRC homology 1 (SH1) domain, a SRC homology 2 (SH2) domain, or an SH1 domain and an SH2 domain.
[0266] Embodiment 63. The chimeric inhibitory receptor of embodiment 58, wherein the enzymatic inhibitory domain comprises a ZAP70 protein with a kinase domain deletion.
[0267] Embodiment 64. The chimeric inhibitory receptor of embodiment 58, wherein the enzymatic inhibitory domain comprises a mutant ZAP70 protein with a Tyr492Phe amino acid substitution, a Tyr493Phe amino acid substitution, or a Tyr492Phe amino acid substitution and a Tyr493Phe amino acid substitution.
[0268] Embodiment 65. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain is derived from RasGAP.
[0269] Embodiment 66. The chimeric inhibitory receptor of any one of embodiments 1-43, wherein the enzymatic inhibitory domain comprises one or more modifications that modulate basal inhibition.
[0270] Embodiment 67. The chimeric inhibitory receptor of embodiment 65, wherein the one or more modifications reduce basal inhibition.
[0271] Embodiment 68. The chimeric inhibitory receptor of embodiment 65, wherein the one or more modifications increase basal inhibition.
[0272] Embodiment 69. The chimeric inhibitory receptor of any one of embodiments 1-68, wherein the enzymatic inhibitory domain inhibits immune receptor activation upon recruitment of the chimeric inhibitory receptor proximal to an immune receptor.
[0273] Embodiment 70. The chimeric inhibitory receptor of any one of embodiments 1-69, wherein the immune receptor is a chimeric immune receptor.
[0274] Embodiment 71. The chimeric inhibitory receptor of embodiment 70, wherein the immune receptor is a chimeric antigen receptor.
[0275] Embodiment 72. The chimeric inhibitory receptor of any one of embodiments 1-69, wherein the immune receptor is a naturally-occurring immune receptor.
[0276] Embodiment 73. The chimeric inhibitory receptor of embodiment 72, wherein the immune receptor is a naturally-occurring antigen receptor.
[0277] Embodiment 74. The chimeric inhibitory receptor of any one of embodiments 1-69, wherein the immune receptor is selected from the group consisting of: a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
[0278] Embodiment 75. The chimeric inhibitory receptor of any one of embodiments 1-73, wherein the immune receptor is a T cell receptor.
[0279] Embodiment 76. A nucleic acid encoding the chimeric inhibitory receptor of any one of embodiments 1-75.
[0280] Embodiment 77. A vector comprising the nucleic acid of embodiment 76.
[0281] Embodiment 78. A genetically engineered cell comprising the nucleic acid of embodiment 76.
[0282] Embodiment 79. A genetically engineered cell comprising the vector of embodiment 77.
[0283] Embodiment 80. A genetically engineered cell expressing the chimeric inhibitory receptor of any one of embodiments 1-75.
[0284] Embodiment 81. A genetically engineered cell expressing a chimeric inhibitory receptor, wherein the chimeric inhibitory receptor comprises:
[0285] an extracellular ligand binding domain;
[0286] a membrane localization domain, wherein the membrane localization domain comprises a transmembrane domain; and
[0287] an enzymatic inhibitory domain, wherein the inhibitory domain inhibits immune receptor activation when proximal to an immune receptor.
[0288] Embodiment 82. The engineered cell of any one of embodiments 78-81, wherein the cell further comprises an immune receptor.
[0289] Embodiment 83. The engineered cell of embodiment 82, wherein the immune receptor is a chimeric immune receptor.
[0290] Embodiment 84. The engineered cell of embodiment 83, wherein the immune receptor is a chimeric antigen receptor.
[0291] Embodiment 85. The engineered cell of embodiment 82, wherein the immune receptor is a naturally-occurring immune receptor.
[0292] Embodiment 86. The engineered cell of embodiment 85, wherein the immune receptor is a naturally-occurring antigen receptor.
[0293] Embodiment 87. The engineered cell of embodiment 82, wherein the immune receptor is selected from the group consisting of: a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activated receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor.
[0294] Embodiment 88. The engineered cell of any one of embodiments 82-87, wherein the chimeric inhibitory receptor inhibits immune receptor activation upon ligand binding.
[0295] Embodiment 89. The engineered cell of any one of embodiments 82-88, wherein the ligand is a cell surface ligand.
[0296] Embodiment 90. The engineered cell of embodiment 89, wherein the cell surface ligand is expressed on a cell that further expresses a cognate immune receptor ligand.
[0297] Embodiment 91. The engineered cell of embodiment 90, wherein ligand binding to the chimeric inhibitory receptor and cognate immune receptor ligand binding to the immune receptor localizes the chimeric inhibitory receptor proximal to the immune receptor.
[0298] Embodiment 92. The engineered cell of embodiment 91, wherein localization of the chimeric inhibitory receptor proximal to the immune receptor inhibits immune receptor activation.
[0299] Embodiment 93. The engineered cell of any one of embodiments 88-93, wherein the cell is a T cell.
[0300] Embodiment 94. The engineered cell of embodiment 93, wherein the immune receptor is a T cell receptor.
[0301] Embodiment 95. The engineered cell of embodiment 94, wherein immune receptor activation is T cell activation.
[0302] Embodiment 96. The engineered cell of any one of embodiments 78-92, wherein the cell is an immunomodulatory cell.
[0303] Embodiment 97. The engineered cell of embodiment 96, wherein the immunomodulatory cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.
[0304] Embodiment 98. The engineered cell of of any one of embodiments 78-97, wherein the cell is autologous.
[0305] Embodiment 99. The engineered cell of of any one of embodiments 78-97, wherein the cell is allogeneic.
[0306] Embodiment 100. A pharmaceutical composition comprising the engineered cell of any one of embodiments 78-99 and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or combination thereof.
[0307] Embodiment 101. A method of inhibiting immune receptor activation, comprising:
[0308] contacting the engineered cell of any one of embodiments 78-99 or the pharmaceutical composition of embodiment 100 with a cognate ligand under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand,
[0309] wherein, when localized proximal to an immune receptor expressed on a cell membrane of the engineered cell, the chimeric inhibitory inhibits immune receptor activation.
[0310] Embodiment 102. A method for reducing an immune response, comprising:
[0311] administering the engineered cell of any one of embodiments 78-99 or the pharmaceutical composition of embodiment 100 to a subject in need of such treatment.
[0312] Embodiment 103. A method of preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, comprising:
[0313] administering the engineered cell of any one of embodiments 78-99 or the pharmaceutical composition of embodiment 100 to a subject in need of such treatment.
[0314] Embodiment 104. A method of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, comprising:
[0315] contacting the engineered cell of any one embodiments 78-99 or the pharmaceutical composition of embodiment 100 with a cognate ligand of the chimeric inhibitory receptor under conditions suitable for the chimeric inhibitory receptor to bind the cognate ligand,
[0316] wherein upon binding of the ligand to the chimeric inhibitory receptor, the enzymatic inhibitory domain prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.
[0317] Embodiment 105. A method for treating an autoimmune disease or disease treatable by reducing an immune response comprising:
[0318] administering the engineered cell of any one of embodiments 78-99 or the pharmaceutical composition of embodiment 100 to a subject in need of such treatment.
EXAMPLES
[0319] The following are examples of methods and compositions of the present disclosure. It is understood that various other embodiments may be practiced, given the general description provided herein.
[0320] Below are examples of specific embodiments for carrying out the claimed subject matter of the present disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1: Inhibition by Enzymatic Inhibitory Domain (EID)-Containing CAR CAR-T and K562 Co-Culture Methods
Lentiviral Production:
[0321] Lentivirus was produced using: Lenti-X 293T packaging cell line (Clontech, Cat #632180); LX293T Complete growth medium, without antibiotics; DMEM, hi-glucose; 1 mM Sodium Pyruvate; 10% FBS, heat-inactivated; Opti-Mem I Reduced Serum Media (Gibco/Thermo Fisher; Cat #31985); FuGene HD (Promega, Cat #E2311); Envelope, Packaging, and Transfer Vector plasmids; VSV-G-pseudotyped envelope vector (pMD2.G); Packaging vector that contains Gag, Pol, Rev, and Tat that can be used with 2nd and 3rd generation transfer vectors (psMAX2). 293T(FT) cells from 90% confluent 10 cm dishes were lifted and dispensed at 1:3 dilution late in the afternoon the day before transfection and incubated cells at normal overnight at 37.degree. C., 5% CO2 (cells should be 60-85% confluent the next day at time of transfection).
[0322] A transfection reaction was prepped for each 10 cm dish according to the protocol below:
[0323] 1. Prep transfection reaction for each 10 cm dish in a separate 1.7 mL tube.
[0324] 2. Add 900 uL Opti-Mem I at RT.
[0325] 3. Add 9 ug vector backbone (containing gene of interest) per reaction.
[0326] 4. Add 8 ug packaging vector per reaction.
[0327] 5. Add 1 ug envelope vector per reaction (pMD2.G).
[0328] 6. Mix thoroughly by quickly vortexing for 3 seconds.
[0329] 7. Add 55 uL Fugene HD per reaction.
[0330] 8. Mix by quickly pipetting up and down 20-30 times.
[0331] 9. Let sit at RT for 10 min (allowing DNA complexes to form).
[0332] 10. Slowly add mixture in dropwise manner around the dish, then mix by gently rocking back-forth and up-down for 5-10 seconds (do not swirl).
[0333] 11. Place dish into virus incubator.
[0334] Viral supernatants were harvested on days 2 and 3 using a serological pipette. Cellular debris was removed using a Millipore steriflip 0.45 um filters. A Lenti-X Concentrator (Cat. Nos. 631231 & 631232) was used according to the protocol: 1) Combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant. Mix by gentle inversion; 2) Incubate mixture on ice or at 4.degree. C. for 30 minutes to overnight; (3) Centrifuge sample at 1,500.times.g for 45 minutes at 4.degree. C.; (4) Carefully remove and discard supernatant, taking care not to disturb the pellet; (5) Gently resuspend the pellet in 1/10 to 1/100th of the original volume using sterile PBS+0.1% BSA.
Transduction and Expansion
[0335] Primary T cells were isolated from human donor PBMCs and frozen. On Day 1, 1.times.10.sup.6 purified CD4+/CD8+ T-cells were thawed and stimulated with 3.times.10.sup.6 Human T-Activator CD3/CD28 Dynabeads, then cultured in 1 mL Optimizer CTS T-cell expansion media (Gibco) with 0.2 .mu.g/mL IL-2. On Day 2, cells were co-transduced with a lentivirus (see production methods above) encoding an activating CAR (aCAR) and/or a lentivirus encoding an inhibitory CAR (iCAR) to produce aCAR+, iCAR+, and aCAR+/iCAR+(dual+) T cells (100K each construct, as quantified by GoStix (Tekara)). Each CAR was under control of a constitutive SFFV promoter. The various aCAR and iCAR constructs and associated CAR domains are described below in Table A and the full coding sequences provided in Table C. On Day 3, Dynabeads were removed by magnet. T-cells were counted and passaged (0.5.times.10.sup.6 cells/mL). During subsequent expansion, cells were passaged every two days (0.5.times.10.sup.6 cells/mL).
TABLE-US-00009 TABLE A CAR Constructs Construct Promoter Signal seq. Tag scFV Hinge TM IC #1 Marker aCAR SFFV CD8 anti-CD20 MYC-CD8 CD28 CD28- CD3zeta iCAR17 SFFV IgGkappa FLAG anti-CD19 CD8 PAG Csk iCAR25 SFFV IgGkappa FLAG anti-CD19 CD8 CD28 Csk 2A-PuroR iCAR26 SFFV IgGkappa FLAG anti-CD19 CD8 CD28 Csk_SH3 2A-PuroR iCAR30 SFFV IgGkappa FLAG anti-CD19 CD8 LAT Csk 2A-PuroR iCAR31 SFFV IgGkappa Flag anti-CD19 CD8 PAG Csk 2A-PuroR
Co-Culture Assay
[0336] On Day 7, an aliquot of each cell population was stained with PE conjugated anti-MYC and BV421 conjugated anti-FLAG antibodies (corresponding to aCAR and iCAR, respectively), and their transgene expression quantified using an LX CytoFlex Flow Cytometry machine. On Day 8, T-cells were counted and distributed into a 96-well plate for co-culture assays, with each well containing 5.times.10.sup.5 K562 target cells either engineered to co-express aCAR target CD20 and iCAR target CD19 or engineered to express CD20 alone and stained with CellTrace Violet dye (Invitrogen) and 5.times.10.sup.5 aCAR+ or dual+ T-cells. Co-cultures incubated (37.degree., 5% CO.sub.2) for 40 hrs. On Day 10, cells in co-cultures were stained with NIR viability dye (Biolegend) and the number of live target cells was quantified using a CytoFlex LX flow cytometer. Killing efficiencies for each engineered CAR-T cell population were calculated as the ratio of surviving wild type K562 relative to each of the CD20-expressing K562 target cell lines. Normalized killing efficiencies were calculated as the ratio of CAR-T killing efficiencies for dual (CD20+CD19+) vs single (CD20+ only) antigen target cells.
Enzymatic Inhibitory Domain Containing CAR Results
[0337] Inhibition of T cell signaling by a CAR containing an enzymatic inhibitory domain (EID) was assessed. The general strategy is schematized in FIGS. 1-3 showing inhibition of signaling mediated by EID-containing chimeric receptors when the receptor engages a cognate ligand expressed on a target cell.
[0338] A system for assessing inhibition by EID-containing chimeric receptors was established. FIG. 4 schematizes the system where k562 target cells were engineered to express a cognate antigen for an aCAR (CD20) or engineered to express both the cognate antigen for the aCAR (CD20) and a cognate antigen for an iCAR (CD19). The system examined assessed the ability of an anti-CD19 iCAR including a CSK domain as the EID domain to inhibit signaling of an aCAR including a CD28-CD3.zeta. intracellular signaling domain. FIG. 5 provides representative flow-cytometry plots demonstrating the iCAR construct anti-CD19_scFv-Csk fusions was expressed at levels detectable above unmodified cells following transduction of CD4+ and CD8+ T cells without subsequent enrichment. Importantly, T cells demonstrated co-expression of both iCAR and aCAR constructs following lentiviral co-transduction (FIG. 5, bottom right). Expression profiles for the various constructs examined was assessed by flow-cytometry and presented in FIG. 6 demonstrating expression of the aCAR and iCAR constructs. Shown is: aCAR+=cells that express the aCAR (w/ and w/out iCAR) [first column]; iCAR+=cells that express the iCAR (w/ and w/out the aCAR) [second column]; and dual+=cells that express both the aCAR and iCAR [third column]. Importantly, a comparison of the aCAR+ population (first column) and dual+ population (third column) demonstrates the majority of the cells expressing an aCAR are dual+(i.e., also express an iCAR), indicating minimal residual aCAR-only cells (i.e., express only an aCAR) were present that would not be inhibited by a functional iCAR.
[0339] The iCAR constructs were then assessed for their ability to inhibit signaling. As shown in FIG. 7, while transduction with an aCAR construct only led to efficient target cell killing (FIG. 7, left column; represented as ratio of killing CD19/CD20 targets cells to CD20-only target cells), co-transduction of T cells with an iCAR possessing a CSK enzymatic inhibitory domain (iCAR31) led to an .about.50% reduction in killing efficiency (FIG. 7, middle column). Co-transduction of T cells with an iCAR possessing a CSK enzymatic inhibitory domain with a deletion in the CSK SH3 domain (iCAR26) did not demonstrate inhibition (FIG. 7, right column). Accordingly, the data demonstrate a CAR containing an enzymatic inhibitory domain was capable of inhibiting cellular signaling mediated by an activating CAR in a ligand-specific manner.
Example 2: Assessment of Enzymatic Inhibitory Domain (EID)-Containing CARs
CAR-T and K562 Co-Culture Methods
Lentiviral Production:
[0340] Lentivirus is produced using: Lenti-X 293T packaging cell line (Clontech, Cat #632180); LX293T Complete growth medium, without antibiotics; DMEM, hi-glucose; 1 mM Sodium Pyruvate; 10% FBS, heat-inactivated; Opti-Mem I Reduced Serum Media (Gibco/Thermo Fisher; Cat #31985); FuGene HD (Promega, Cat #E2311); Envelope, Packaging, and Transfer Vector plasmids; VSV-G-pseudotyped envelope vector (pMD2.G); Packaging vector that contains Gag, Pol, Rev, and Tat that can be used with 2nd and 3rd generation transfer vectors (psMAX2). 293T(FT) cells from 90% confluent 10 cm dishes are lifted and dispensed at 1:3 dilution late in the afternoon the day before transfection and cells are incubated at normal overnight at 37.degree. C., 5% CO.sub.2 (cells should be 60-85% confluent the next day at time of transfection).
[0341] A transfection reaction is prepped for each 10 cm dish according to the protocol below:
[0342] 1. Prep transfection reaction for each 10 cm dish in a separate 1.7 mL tube.
[0343] 2. Add 900 uL Opti-Mem I at RT.
[0344] 3. Add 9 ug vector backbone (containing gene of interest) per reaction.
[0345] 4. Add 8 ug packaging vector per reaction.
[0346] 5. Add 1 ug envelope vector per reaction (pMD2.G).
[0347] 6. Mix thoroughly by quickly vortexing for 3 seconds.
[0348] 7. Add 55 uL Fugene HD per reaction.
[0349] 8. Mix by quickly pipetting up and down 20-30 times.
[0350] 9. Let sit at RT for 10 min (allowing DNA complexes to form).
[0351] 10. Slowly add mixture in dropwise manner around the dish, then mix by gently rocking back-forth and up-down for 5-10 seconds (do not swirl).
[0352] 11. Place dish into virus incubator.
[0353] Viral supernatants are harvested on days 2 and 3 using a serological pipette. Cellular debris are removed using a Millipore steriflip 0.45 um filters. A Lenti-X Concentrator (Cat. Nos. 631231 & 631232) is used according to the protocol: 1) Combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant. Mix by gentle inversion; 2) Incubate mixture on ice or at 4.degree. C. for 30 minutes to overnight; (3) Centrifuge sample at 1,500.times.g for 45 minutes at 4.degree. C.; (4) Carefully remove and discard supernatant, taking care not to disturb the pellet; (5) Gently resuspend the pellet in 1/10 to 1/100th of the original volume using sterile PBS+0.1% BSA.
Transduction and Expansion
[0354] Primary T cells are isolated from human donor PBMCs and frozen. On Day 1, 1.times.10.sup.6 purified CD4+/CD8+ T-cells are thawed and stimulated with 3.times.10.sup.6 Human T-Activator CD3/CD28 Dynabeads, then cultured in 1 mL Optimizer CTS T-cell expansion media (Gibco) with 0.2 .mu.g/mL IL-2. On Day 2, cells are co-transduced with a lentivirus (see production methods above) encoding an activating CAR (aCAR) and/or a lentivirus encoding an inhibitory CAR (iCAR) to produce aCAR+, iCAR+, and aCAR+/iCAR+(dual+) T cells (100K each construct, as quantified by GoStix (Tekara)). Each CAR is under control of a constitutive SFFV promoter. The various aCAR and iCAR constructs and associated CAR domains are described below in Table B. On Day 3, Dynabeads are removed by magnet. T-cells are counted and passaged (0.5.times.10.sup.6 cells/mL). During subsequent expansion, cells are passaged every two days (0.5.times.10.sup.6 cells/mL).
Co-Culture Assay
[0355] On Day 7, an aliquot of each cell population is stained with PE conjugated anti-MYC and BV421 conjugated anti-FLAG antibodies (corresponding to aCAR and iCAR, respectively), and their transgene expression is quantified using an LX CytoFlex Flow Cytometry machine. On Day 8, T-cells are counted and distributed into a 96-well plate for co-culture assays, with each well containing 5.times.10.sup.5 K562 target cells either engineered to co-express aCAR target CD20 and iCAR target CD19 or engineered to express CD20 alone and stained with CellTrace Violet dye (Invitrogen) and 5.times.10.sup.5 aCAR+ or dual+ T-cells. Co-cultures are incubated (37.degree., 5% CO.sub.2) for 40 hrs. On Day 10, cells in co-cultures are stained with NIR viability dye (Biolegend) and the number of live target cells is quantified using a CytoFlex LX flow cytometer. Killing efficiencies for each engineered CAR-T cell population are calculated as the ratio of surviving wild type K562 relative to each of the CD20-expressing K562 target cell lines. Normalized killing efficiencies are calculated as the ratio of CAR-T killing efficiencies for dual (CD20+CD19+) vs single (CD20+ only) antigen target cells.
Enzymatic Inhibitory Domain Containing CAR Assessment Results
[0356] Inhibition of T cell signaling by a CAR containing an enzymatic inhibitory domain (EID) is assessed. The assessment strategy follows that described in Example 1. Engineered T cells expressing an aCAR alone or co-expressing an aCAR and iCAR are assessed for cytotoxicity, cytokine release, expression of activation-associated markers when co-cultured with engineered target cells expressing cognate antigens recognized by the iCAR, aCAR, both, or neither. Exemplary constructs assessed are described in Table B. Also assessed are aCARs targeting tumor-associated antigens in combination with iCARs targeting antigens generally expressed on healthy tissue and/or cells.
[0357] Flow-cytometry analysis of engineered T cells demonstrates co-expression of aCAR and iCAR constructs. The various iCAR constructs are then assessed for their ability to inhibit signaling. Results demonstrate CARs containing an enzymatic inhibitory domain are capable of inhibiting cellular signaling of an activating CAR in a ligand-specific manner, including determining those iCAR features (e.g., EIDs, additional domains, domain organizations, etc.) demonstrating the most robust signaling inhibition and/or ligand-specificity.
TABLE-US-00010 TABLE B Candidate iCAR Constructs Signal Epitope hinge/ TM Construct Sequence Tag scFv linker Domain Linker Intracellular Domain 2A Marker SB00628 CD8 aCD19 CD8 CD28 Csk SB00629 CD8 aCD19 CD8 CD28 Csk deltaSH3 SB00655 CD8 aCD19 CD8 LAT (1-33) Csk deltaSH3 SB00656 CD8 aCD19 CD8 PAG (1-46) Csk deltaSH3 SB01012 mIgK FLAG aCD19 CD8 CD28 Csk deltaSH3 SB01016 mIgK FLAG aCD19 CD8 LAT (1-33) Csk deltaSH3 SB01017 mIgK FLAG aCD19 CD8 PAG (1-46) Csk deltaSH3 SB01025 mIgK FLAG aCD19 CD8 CD28 Csk T2A PuroR SB01026 mIgK FLAG aCD19 CD8 CD28 Csk deltaSH3 T2A PuroR SB01030 mIgK FLAG aCD19 CD8 LAT (1-33) Csk deltaSH3 T2A PuroR SB01031 mIgK FLAG aCD19 CD8 PAG (1-46) Csk deltaSH3 T2A PuroR SB01871 mIgK FLAG aCD19 CD8 mLAT (G4S)3 Csk deltaSH3 T2A GFP SB01872 mIgK FLAG aCD19 CD8 mLAT (G4S)3 SHP-1 T2A GFP SB01873 mIgK FLAG aCD19 CD8 mLAT (G4S)3 SHP-1 PTP domain T2A GFP SB01874 mIgK FLAG aCD19 CD8 mLAT (G4S)3 SHP-2 T2A GFP SB01875 mIgK FLAG aCD19 CD8 mLAT(ca) (G4S)3 SHP-1 T2A GFP SB01876 mIgK FLAG aCD19 CD8 LAX (G4S)3 SHP-1 T2A GFP SB01877 CD8 FLAG aCD19 CD8 mLAT (G4S)3 SHP-1 PTP domain T2A GFP SB01878 mIgK FLAG aCD19 CD8 CD28 (G4S)3 SHP-1 T2A GFP SB01879 mIgK FLAG aCD19 CD8 CD3z (G4S)3 SHP-1 T2A GFP SB01881 mIgK FLAG aCD19 CD8 CD45 CD45 T2A GFP SB01884 mIgK FLAG aCD19 CD8 CD8 (G4S)3 PTEN T2A GFP SB01885 mIgK FLAG aCD19 CD8 CD8 (G4S)3 PTP-MEG1 T2A GFP SB01886 mIgK FLAG aCD19 CD8 CD8 (G4S)3 PTPN22 T2A GFP SB02018 GM-CSF FLAG aCD19 CD8 LAT(1-33) (G4S)3 Csk T2A GFP SB02033 mIgK FLAG aCD19 CD8 CD8 (G4S)3 Zap70, SH2 domains T2A GFP (G4S)3 1 and 2 only SB02034 mIgK FLAG aCD19 CD8 CD8 (G4S)3 Zap70 delta kinase T2A GFP SB02035 mIgK FLAG aCD19 CD8 CD8 (G4S)3 Zap70 Y492F Y493F T2A GFP SB02162 mIgK FLAG aCD19 CD8 CD8 (G4S)3 SHIP-1 PTP domain
Other Embodiments
[0358] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
[0359] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS
[0360] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
[0361] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
[0362] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
[0363] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
[0364] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0365] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0366] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[0367] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
[0368] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of" and "consisting essentially of" the feature described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."
ADDITIONAL SEQUENCES
[0369] Certain additional sequences for vectors, cassettes and protein domains referred to herein are described below and referred to by SEQ ID NO.
TABLE-US-00011 TABLE C Additional Sequences Amino Acid Sequence SEQ ID NO: Description MALPVTALLLPLALLLHAARP 81 CD8 signal sequence METDTLLLWVLLLWVPGSTGAGGS 82 mIgK signal sequence MLLLVTSLLLCELPHPAFLLIP 83 GM-CSF signal sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 84 aCD19 scFv QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKS QVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWG QGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTS RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG NTLPYTFGGGTKLEIT MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGA 85 aCAR full coding sequence SVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGFIT PTTGYPEYNQKFKDRVTMTADKSTSTAYMELSSLRSE DTAVYYCARRKVGKGVYYALDYWGQGTTVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRA SGNIHNYLAWYQQKPGKVPKLLIYNTKTLADGVPSRFS GSGSGTDYTLTISSLQPEDVATYYCQHFWSSPWTFGGG TKVEIKEQKLISEEDLNGAATTTPAPRPPTPAPTIALQPL SLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR* METDTLLLWVLLLWVPGSTGAGGSDYKDDDDKGGSE 86 iCAR17 full coding sequence VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITTTTPAPRPPTPAPTIALQPLSLRPE ACRPAAGGAVHTRGLDFACDGPAGSLLGSGQMQITLW GSLAAVAIFFVITFLIFLCSSCDREKKPRSAIQAAWPSGT ECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYK AKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGK ITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGK VEHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADG LCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQ TIGKGEFGDVMLGDYRGNKVAVKCIKNDATAQAFLAE ASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGS LVDYLRSRGRSVLGGDCLLKFSLDVCEAMEYLEGNNF VHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGK LPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRV PYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNC WHLDAAMRPSFLQLREQLEHIKTHELHL* METDTLLLWVLLLWVPGSTGAGGSDYKDDDDKGGSE 87 iCAR25 full coding sequence VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITTTTPAPRPPTPAPTIALQPLSLRPE ACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSL LVTVAFIIFWVSAIQAAWPSGTECIAKYNFHGTAEQDLP FCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQ KREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLF LVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDE EVYFENLMQLVEHYTSDADGLCTRLIKPKVMEGTVAA QDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYR GNKVAVKCIKNDATAQAFLAEASVMTQLRHSNLVQLL GVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGD CLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDN VAKVSDFGLTKEASSTQDTGKLPVKWTAPEALREKKF STKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKG YKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLRE QLEHIKTHELHLEGRGSLLTCGDVEENPGPMTEYKPTV RLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESV EAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPK EPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAG VPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCM TRKPGA* METDTLLLWVLLLWVPGSTGAGGSDYKDDDDKGGSE 88 iCAR26 full coding sequence VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITTTTPAPRPPTPAPTIALQPLSLRPE ACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSL LVTVAFIIFWVVKAGTKLSLMPWFHGKITREQAERLLY PPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHA SKLSIDEEVYFENLMQLVEHYTSDADGLCTRLIKPKVM EGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDV MLGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRH SNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGR SVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNV LVSEDNVAKVSDFGLTKEASSTQDTGKLPVKWTAPEA LREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVV PRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRP SFLQLREQLEHIKTHELHLEGRGSLLTCGDVEENPGPM TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVD PDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAV WTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGL LAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVE AAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEG PRTWCMTRKPGA* METDTLLLWVLLLWVPGSTGAGGSDYKDDDDKGGSE 89 iCAR30 full coding sequence VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITTTTPAPRPPTPAPTIALQPLSLRPE ACRPAAGGAVHTRGLDFACDEEAILVPCVLGLLLLPIL AMLMALCVHCHRLPSAIQAAWPSGTECIAKYNFHGTA EQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPA NYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPP ETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHAS KLSIDEEVYFENLMQLVEHYTSDADGLCTRLIKPKVME GTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVM LGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRHS NLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRS VLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVL VSEDNVAKVSDFGLTKEASSTQDTGKLPVKWTAPEAL REKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVP RVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPS FLQLREQLEHIKTHELHLEGRGSLLTCGDVEENPGPMT EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDP DRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVW TTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLA PHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAA ERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPR TWCMTRKPGA* METDTLLLWVLLLWVPGSTGAGGSDYKDDDDKGGSE 90 iCAR31 full coding sequence VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITTTTPAPRPPTPAPTIALQPLSLRPE ACRPAAGGAVHTRGLDFACDGPAGSLLGSGQMQITLW GSLAAVAIFFVITFLIFLCSSCDREKKPRSAIQAAWPSGT ECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYK AKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGK ITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGK VEHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADG LCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQ TIGKGEFGDVMLGDYRGNKVAVKCIKNDATAQAFLAE ASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGS LVDYLRSRGRSVLGGDCLLKFSLDVCEAMEYLEGNNF VHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGK LPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRV PYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNC WHLDAAMRPSFLQLREQLEHIKTHELHLEGRGSLLTCG DVEENPGPMTEYKPTVRLATRDDVPRAVRTLAAAFAD YPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVA DDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLG SAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTV TADVEVPEGPRTWCMTRKPGA*
Sequence CWU
1
1
901449PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 1Ser Ala Ile Gln Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile
Ala Lys1 5 10 15Tyr Asn
Phe His Gly Thr Ala Glu Gln Asp Leu Pro Phe Cys Lys Gly 20
25 30Asp Val Leu Thr Ile Val Ala Val Thr
Lys Asp Pro Asn Trp Tyr Lys 35 40
45Ala Lys Asn Lys Val Gly Arg Glu Gly Ile Ile Pro Ala Asn Tyr Val 50
55 60Gln Lys Arg Glu Gly Val Lys Ala Gly
Thr Lys Leu Ser Leu Met Pro65 70 75
80Trp Phe His Gly Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu
Leu Tyr 85 90 95Pro Pro
Glu Thr Gly Leu Phe Leu Val Arg Glu Ser Thr Asn Tyr Pro 100
105 110Gly Asp Tyr Thr Leu Cys Val Ser Cys
Asp Gly Lys Val Glu His Tyr 115 120
125Arg Ile Met Tyr His Ala Ser Lys Leu Ser Ile Asp Glu Glu Val Tyr
130 135 140Phe Glu Asn Leu Met Gln Leu
Val Glu His Tyr Thr Ser Asp Ala Asp145 150
155 160Gly Leu Cys Thr Arg Leu Ile Lys Pro Lys Val Met
Glu Gly Thr Val 165 170
175Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys
180 185 190Glu Leu Lys Leu Leu Gln
Thr Ile Gly Lys Gly Glu Phe Gly Asp Val 195 200
205Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala Val Lys Cys
Ile Lys 210 215 220Asn Asp Ala Thr Ala
Gln Ala Phe Leu Ala Glu Ala Ser Val Met Thr225 230
235 240Gln Leu Arg His Ser Asn Leu Val Gln Leu
Leu Gly Val Ile Val Glu 245 250
255Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly Ser
260 265 270Leu Val Asp Tyr Leu
Arg Ser Arg Gly Arg Ser Val Leu Gly Gly Asp 275
280 285Cys Leu Leu Lys Phe Ser Leu Asp Val Cys Glu Ala
Met Glu Tyr Leu 290 295 300Glu Gly Asn
Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val Leu305
310 315 320Val Ser Glu Asp Asn Val Ala
Lys Val Ser Asp Phe Gly Leu Thr Lys 325
330 335Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro
Val Lys Trp Thr 340 345 350Ala
Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys Ser Asp Val 355
360 365Trp Ser Phe Gly Ile Leu Leu Trp Glu
Ile Tyr Ser Phe Gly Arg Val 370 375
380Pro Tyr Pro Arg Ile Pro Leu Lys Asp Val Val Pro Arg Val Glu Lys385
390 395 400Gly Tyr Lys Met
Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr Glu 405
410 415Val Met Lys Asn Cys Trp His Leu Asp Ala
Ala Met Arg Pro Ser Phe 420 425
430Leu Gln Leu Arg Glu Gln Leu Glu His Ile Lys Thr His Glu Leu His
435 440 445Leu2380PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Val Lys Ala Gly Thr Lys Leu Ser Leu Met Pro Trp Phe His Gly Lys1
5 10 15Ile Thr Arg Glu Gln Ala
Glu Arg Leu Leu Tyr Pro Pro Glu Thr Gly 20 25
30Leu Phe Leu Val Arg Glu Ser Thr Asn Tyr Pro Gly Asp
Tyr Thr Leu 35 40 45Cys Val Ser
Cys Asp Gly Lys Val Glu His Tyr Arg Ile Met Tyr His 50
55 60Ala Ser Lys Leu Ser Ile Asp Glu Glu Val Tyr Phe
Glu Asn Leu Met65 70 75
80Gln Leu Val Glu His Tyr Thr Ser Asp Ala Asp Gly Leu Cys Thr Arg
85 90 95Leu Ile Lys Pro Lys Val
Met Glu Gly Thr Val Ala Ala Gln Asp Glu 100
105 110Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys Glu
Leu Lys Leu Leu 115 120 125Gln Thr
Ile Gly Lys Gly Glu Phe Gly Asp Val Met Leu Gly Asp Tyr 130
135 140Arg Gly Asn Lys Val Ala Val Lys Cys Ile Lys
Asn Asp Ala Thr Ala145 150 155
160Gln Ala Phe Leu Ala Glu Ala Ser Val Met Thr Gln Leu Arg His Ser
165 170 175Asn Leu Val Gln
Leu Leu Gly Val Ile Val Glu Glu Lys Gly Gly Leu 180
185 190Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly Ser
Leu Val Asp Tyr Leu 195 200 205Arg
Ser Arg Gly Arg Ser Val Leu Gly Gly Asp Cys Leu Leu Lys Phe 210
215 220Ser Leu Asp Val Cys Glu Ala Met Glu Tyr
Leu Glu Gly Asn Asn Phe225 230 235
240Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Ser Glu Asp
Asn 245 250 255Val Ala Lys
Val Ser Asp Phe Gly Leu Thr Lys Glu Ala Ser Ser Thr 260
265 270Gln Asp Thr Gly Lys Leu Pro Val Lys Trp
Thr Ala Pro Glu Ala Leu 275 280
285Arg Glu Lys Lys Phe Ser Thr Lys Ser Asp Val Trp Ser Phe Gly Ile 290
295 300Leu Leu Trp Glu Ile Tyr Ser Phe
Gly Arg Val Pro Tyr Pro Arg Ile305 310
315 320Pro Leu Lys Asp Val Val Pro Arg Val Glu Lys Gly
Tyr Lys Met Asp 325 330
335Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr Glu Val Met Lys Asn Cys
340 345 350Trp His Leu Asp Ala Ala
Met Arg Pro Ser Phe Leu Gln Leu Arg Glu 355 360
365Gln Leu Glu His Ile Lys Thr His Glu Leu His Leu 370
375 3803595PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 3Met Val Arg Trp Phe
His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr1 5
10 15Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe
Leu Ala Arg Pro Ser 20 25
30Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln
35 40 45Val Thr His Ile Arg Ile Gln Asn
Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55
60Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr65
70 75 80Gln Gln Gln Gly Val
Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85
90 95Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser
Glu Arg Trp Tyr His 100 105
110Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly
115 120 125Glu Pro Trp Thr Phe Leu Val
Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135
140Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly
Ser145 150 155 160Pro Leu
Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr
165 170 175Thr Val Gly Gly Leu Glu Thr
Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185
190His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe
Val Tyr 195 200 205Leu Arg Gln Pro
Tyr Tyr Ala Thr Arg Val Asn Ala Ala Asp Ile Glu 210
215 220Asn Arg Val Leu Glu Leu Asn Lys Lys Gln Glu Ser
Glu Asp Thr Ala225 230 235
240Lys Ala Gly Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val
245 250 255Lys Asn Leu His Gln
Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly 260
265 270Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His
Ser Arg Val Ile 275 280 285Leu Gln
Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala 290
295 300Asn Tyr Ile Lys Asn Gln Leu Leu Gly Pro Asp
Glu Asn Ala Lys Thr305 310 315
320Tyr Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp
325 330 335Gln Met Ala Trp
Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg 340
345 350Glu Val Glu Lys Gly Arg Asn Lys Cys Val Pro
Tyr Trp Pro Glu Val 355 360 365Gly
Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu 370
375 380His Asp Thr Thr Glu Tyr Lys Leu Arg Thr
Leu Gln Val Ser Pro Leu385 390 395
400Asp Asn Gly Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu
Ser 405 410 415Trp Pro Asp
His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe 420
425 430Leu Asp Gln Ile Asn Gln Arg Gln Glu Ser
Leu Pro His Ala Gly Pro 435 440
445Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile 450
455 460Val Ile Asp Met Leu Met Glu Asn
Ile Ser Thr Lys Gly Leu Asp Cys465 470
475 480Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val Arg
Ala Gln Arg Ser 485 490
495Gly Met Val Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile
500 505 510Ala Gln Phe Ile Glu Thr
Thr Lys Lys Lys Leu Glu Val Leu Gln Ser 515 520
525Gln Lys Gly Gln Glu Ser Glu Tyr Gly Asn Ile Thr Tyr Pro
Pro Ala 530 535 540Met Lys Asn Ala His
Ala Lys Ala Ser Arg Thr Ser Ser Lys His Lys545 550
555 560Glu Asp Val Tyr Glu Asn Leu His Thr Lys
Asn Lys Arg Glu Glu Lys 565 570
575Val Lys Lys Gln Arg Ser Ala Asp Lys Glu Lys Ser Lys Gly Ser Leu
580 585 590Lys Arg Lys
5954272PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 4Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val Lys
Asn Leu1 5 10 15His Gln
Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys Asn Arg 20
25 30Tyr Lys Asn Ile Leu Pro Phe Asp His
Ser Arg Val Ile Leu Gln Gly 35 40
45Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Ile 50
55 60Lys Asn Gln Leu Leu Gly Pro Asp Glu
Asn Ala Lys Thr Tyr Ile Ala65 70 75
80Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln
Met Ala 85 90 95Trp Gln
Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu Val Glu 100
105 110Lys Gly Arg Asn Lys Cys Val Pro Tyr
Trp Pro Glu Val Gly Met Gln 115 120
125Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu His Asp Thr
130 135 140Thr Glu Tyr Lys Leu Arg Thr
Leu Gln Val Ser Pro Leu Asp Asn Gly145 150
155 160Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu
Ser Trp Pro Asp 165 170
175His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe Leu Asp Gln
180 185 190Ile Asn Gln Arg Gln Glu
Ser Leu Pro His Ala Gly Pro Ile Ile Val 195 200
205His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile Val
Ile Asp 210 215 220Met Leu Met Glu Asn
Ile Ser Thr Lys Gly Leu Asp Cys Asp Ile Asp225 230
235 240Ile Gln Lys Thr Ile Gln Met Val Arg Ala
Gln Arg Ser Gly Met Val 245 250
255Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile Ala Gln Phe
260 265 2705597PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Met Thr Ser Arg Arg Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala1
5 10 15Glu Asn Leu Leu Leu Thr
Arg Gly Val Asp Gly Ser Phe Leu Ala Arg 20 25
30Pro Ser Lys Ser Asn Pro Gly Asp Phe Thr Leu Ser Val
Arg Arg Asn 35 40 45Gly Ala Val
Thr His Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp 50
55 60Leu Tyr Gly Gly Glu Lys Phe Ala Thr Leu Ala Glu
Leu Val Gln Tyr65 70 75
80Tyr Met Glu His His Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile
85 90 95Glu Leu Lys Tyr Pro Leu
Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp 100
105 110Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys
Leu Leu Thr Glu 115 120 125Lys Gly
Lys His Gly Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro 130
135 140Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp
Asp Lys Gly Glu Ser145 150 155
160Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile Arg Cys Gln Glu
165 170 175Leu Lys Tyr Asp
Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp 180
185 190Leu Val Glu His Tyr Lys Lys Asn Pro Met Val
Glu Thr Leu Gly Thr 195 200 205Val
Leu Gln Leu Lys Gln Pro Leu Asn Thr Thr Arg Ile Asn Ala Ala 210
215 220Glu Ile Glu Ser Arg Val Arg Glu Leu Ser
Lys Leu Ala Glu Thr Thr225 230 235
240Asp Lys Val Lys Gln Gly Phe Trp Glu Glu Phe Glu Thr Leu Gln
Gln 245 250 255Gln Glu Cys
Lys Leu Leu Tyr Ser Arg Lys Glu Gly Gln Arg Gln Glu 260
265 270Asn Lys Asn Lys Asn Arg Tyr Lys Asn Ile
Leu Pro Phe Asp His Thr 275 280
285Arg Val Val Leu His Asp Gly Asp Pro Asn Glu Pro Val Ser Asp Tyr 290
295 300Ile Asn Ala Asn Ile Ile Met Pro
Glu Phe Glu Thr Lys Cys Asn Asn305 310
315 320Ser Lys Pro Lys Lys Ser Tyr Ile Ala Thr Gln Gly
Cys Leu Gln Asn 325 330
335Thr Val Asn Asp Phe Trp Arg Met Val Phe Gln Glu Asn Ser Arg Val
340 345 350Ile Val Met Thr Thr Lys
Glu Val Glu Arg Gly Lys Ser Lys Cys Val 355 360
365Lys Tyr Trp Pro Asp Glu Tyr Ala Leu Lys Glu Tyr Gly Val
Met Arg 370 375 380Val Arg Asn Val Lys
Glu Ser Ala Ala His Asp Tyr Thr Leu Arg Glu385 390
395 400Leu Lys Leu Ser Lys Val Gly Gln Ala Leu
Leu Gln Gly Asn Thr Glu 405 410
415Arg Thr Val Trp Gln Tyr His Phe Arg Thr Trp Pro Asp His Gly Val
420 425 430Pro Ser Asp Pro Gly
Gly Val Leu Asp Phe Leu Glu Glu Val His His 435
440 445Lys Gln Glu Ser Ile Met Asp Ala Gly Pro Val Val
Val His Cys Ser 450 455 460Ala Gly Ile
Gly Arg Thr Gly Thr Phe Ile Val Ile Asp Ile Leu Ile465
470 475 480Asp Ile Ile Arg Glu Lys Gly
Val Asp Cys Asp Ile Asp Val Pro Lys 485
490 495Thr Ile Gln Met Val Arg Ser Gln Arg Ser Gly Met
Val Gln Thr Glu 500 505 510Ala
Gln Tyr Arg Phe Ile Tyr Met Ala Val Gln His Tyr Ile Glu Thr 515
520 525Leu Gln Arg Arg Ile Glu Glu Glu Gln
Lys Ser Lys Arg Lys Gly His 530 535
540Glu Tyr Thr Asn Ile Lys Tyr Ser Leu Ala Asp Gln Thr Ser Gly Asp545
550 555 560Gln Ser Pro Leu
Pro Pro Cys Thr Pro Thr Pro Pro Cys Ala Glu Met 565
570 575Arg Glu Asp Ser Ala Arg Val Tyr Glu Asn
Val Gly Leu Met Gln Gln 580 585
590Gln Lys Ser Phe Arg 5956708PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 6Tyr Lys Ile Tyr Asp
Leu His Lys Lys Arg Ser Cys Asn Leu Asp Glu1 5
10 15Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys
Gln Leu Met Asn Val 20 25
30Glu Pro Ile His Ala Asp Ile Leu Leu Glu Thr Tyr Lys Arg Lys Ile
35 40 45Ala Asp Glu Gly Arg Leu Phe Leu
Ala Glu Phe Gln Ser Ile Pro Arg 50 55
60Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys Pro Phe Asn Gln65
70 75 80Asn Lys Asn Arg Tyr
Val Asp Ile Leu Pro Tyr Asp Tyr Asn Arg Val 85
90 95Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser
Asn Tyr Ile Asn Ala 100 105
110Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr Ile Ala Ala Gln
115 120 125Gly Pro Arg Asp Glu Thr Val
Asp Asp Phe Trp Arg Met Ile Trp Glu 130 135
140Gln Lys Ala Thr Val Ile Val Met Val Thr Arg Cys Glu Glu Gly
Asn145 150 155 160Arg Asn
Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu Glu Gly Thr Arg
165 170 175Ala Phe Gly Asp Val Val Val
Lys Ile Asn Gln His Lys Arg Cys Pro 180 185
190Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys Lys Glu
Lys Ala 195 200 205Thr Gly Arg Glu
Val Thr His Ile Gln Phe Thr Ser Trp Pro Asp His 210
215 220Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu
Arg Arg Arg Val225 230 235
240Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val Val His Cys Ser
245 250 255Ala Gly Val Gly Arg
Thr Gly Thr Tyr Ile Gly Ile Asp Ala Met Leu 260
265 270Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr
Gly Tyr Val Val 275 280 285Lys Leu
Arg Arg Gln Arg Cys Leu Met Val Gln Val Glu Ala Gln Tyr 290
295 300Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn
Gln Phe Gly Glu Thr305 310 315
320Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu His Asn Met Lys Lys
325 330 335Arg Asp Pro Pro
Ser Glu Pro Ser Pro Leu Glu Ala Glu Phe Gln Arg 340
345 350Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His
Ile Gly Asn Gln Glu 355 360 365Glu
Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile Pro Tyr Asp Tyr 370
375 380Asn Arg Val Pro Leu Lys His Glu Leu Glu
Met Ser Lys Glu Ser Glu385 390 395
400His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser Asp Ser Glu Glu
Pro 405 410 415Ser Lys Tyr
Ile Asn Ala Ser Phe Ile Met Ser Tyr Trp Lys Pro Glu 420
425 430Val Met Ile Ala Ala Gln Gly Pro Leu Lys
Glu Thr Ile Gly Asp Phe 435 440
445Trp Gln Met Ile Phe Gln Arg Lys Val Lys Val Ile Val Met Leu Thr 450
455 460Glu Leu Lys His Gly Asp Gln Glu
Ile Cys Ala Gln Tyr Trp Gly Glu465 470
475 480Gly Lys Gln Thr Tyr Gly Asp Ile Glu Val Asp Leu
Lys Asp Thr Asp 485 490
495Lys Ser Ser Thr Tyr Thr Leu Arg Val Phe Glu Leu Arg His Ser Lys
500 505 510Arg Lys Asp Ser Arg Thr
Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser 515 520
525Val Glu Gln Leu Pro Ala Glu Pro Lys Glu Leu Ile Ser Met
Ile Gln 530 535 540Val Val Lys Gln Lys
Leu Pro Gln Lys Asn Ser Ser Glu Gly Asn Lys545 550
555 560His His Lys Ser Thr Pro Leu Leu Ile His
Cys Arg Asp Gly Ser Gln 565 570
575Gln Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu
580 585 590Thr Glu Glu Val Val
Asp Ile Phe Gln Val Val Lys Ala Leu Arg Lys 595
600 605Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr
Gln Phe Leu Tyr 610 615 620Asp Val Ile
Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln Val Lys Lys625
630 635 640Asn Asn His Gln Glu Asp Lys
Ile Glu Phe Asp Asn Glu Val Asp Lys 645
650 655Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu Gly
Ala Pro Glu Lys 660 665 670Leu
Pro Glu Ala Lys Glu Gln Ala Glu Gly Ser Glu Pro Thr Ser Gly 675
680 685Thr Glu Gly Pro Glu His Ser Val Asn
Gly Pro Ala Ser Pro Ala Leu 690 695
700Asn Gln Gly Ser7057217PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Met Glu Ala Ile Ala Lys Tyr Asp Phe
Lys Ala Thr Ala Asp Asp Glu1 5 10
15Leu Ser Phe Lys Arg Gly Asp Ile Leu Lys Val Leu Asn Glu Glu
Cys 20 25 30Asp Gln Asn Trp
Tyr Lys Ala Glu Leu Asn Gly Lys Asp Gly Phe Ile 35
40 45Pro Lys Asn Tyr Ile Glu Met Lys Pro His Pro Trp
Phe Phe Gly Lys 50 55 60Ile Pro Arg
Ala Lys Ala Glu Glu Met Leu Ser Lys Gln Arg His Asp65 70
75 80Gly Ala Phe Leu Ile Arg Glu Ser
Glu Ser Ala Pro Gly Asp Phe Ser 85 90
95Leu Ser Val Lys Phe Gly Asn Asp Val Gln His Phe Lys Val
Leu Arg 100 105 110Asp Gly Ala
Gly Lys Tyr Phe Leu Trp Val Val Lys Phe Asn Ser Leu 115
120 125Asn Glu Leu Val Asp Tyr His Arg Ser Thr Ser
Val Ser Arg Asn Gln 130 135 140Gln Ile
Phe Leu Arg Asp Ile Glu Gln Val Pro Gln Gln Pro Thr Tyr145
150 155 160Val Gln Ala Leu Phe Asp Phe
Asp Pro Gln Glu Asp Gly Glu Leu Gly 165
170 175Phe Arg Arg Gly Asp Phe Ile His Val Met Asp Asn
Ser Asp Pro Asn 180 185 190Trp
Trp Lys Gly Ala Cys His Gly Gln Thr Gly Met Phe Pro Arg Asn 195
200 205Tyr Val Thr Pro Val Asn Arg Asn Val
210 2158403PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Met Thr Ala Ile Ile Lys
Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr1 5
10 15Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile
Tyr Pro Asn Ile 20 25 30Ile
Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35
40 45Asn Ile Asp Asp Val Val Arg Phe Leu
Asp Ser Lys His Lys Asn His 50 55
60Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys65
70 75 80Phe Asn Cys Arg Val
Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85
90 95Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp
Leu Asp Gln Trp Leu 100 105
110Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys
115 120 125Gly Arg Thr Gly Val Met Ile
Cys Ala Tyr Leu Leu His Arg Gly Lys 130 135
140Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg
Thr145 150 155 160Arg Asp
Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr
165 170 175Tyr Tyr Ser Tyr Leu Leu Lys
Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe
Ser Gly 195 200 205Gly Thr Cys Asn
Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210
215 220Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr225 230 235
240Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu
245 250 255Phe Phe His Lys Gln
Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260
265 270Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu
Glu Thr Ser Glu 275 280 285Lys Val
Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290
295 300Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr
Leu Val Leu Thr Leu305 310 315
320Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr
325 330 335Phe Ser Pro Asn
Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340
345 350Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr
Ser Val Thr Pro Asp 355 360 365Val
Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370
375 380Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu
Asp Gln His Thr Gln Ile385 390 395
400Thr Lys Val9926PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Met Thr Ser Arg Phe Arg
Leu Pro Ala Gly Arg Thr Tyr Asn Val Arg1 5
10 15Ala Ser Glu Leu Ala Arg Asp Arg Gln His Thr Glu
Val Val Cys Asn 20 25 30Ile
Leu Leu Leu Asp Asn Thr Val Gln Ala Phe Lys Val Asn Lys His 35
40 45Asp Gln Gly Gln Val Leu Leu Asp Val
Val Phe Lys His Leu Asp Leu 50 55
60Thr Glu Gln Asp Tyr Phe Gly Leu Gln Leu Ala Asp Asp Ser Thr Asp65
70 75 80Asn Pro Arg Trp Leu
Asp Pro Asn Lys Pro Ile Arg Lys Gln Leu Lys 85
90 95Arg Gly Ser Pro Tyr Ser Leu Asn Phe Arg Val
Lys Phe Phe Val Ser 100 105
110Asp Pro Asn Lys Leu Gln Glu Glu Tyr Thr Arg Tyr Gln Tyr Phe Leu
115 120 125Gln Ile Lys Gln Asp Ile Leu
Thr Gly Arg Leu Pro Cys Pro Ser Asn 130 135
140Thr Ala Ala Leu Leu Ala Ser Phe Ala Val Gln Ser Glu Leu Gly
Asp145 150 155 160Tyr Asp
Gln Ser Glu Asn Leu Ser Gly Tyr Leu Ser Asp Tyr Ser Phe
165 170 175Ile Pro Asn Gln Pro Gln Asp
Phe Glu Lys Glu Ile Ala Lys Leu His 180 185
190Gln Gln His Ile Gly Leu Ser Pro Ala Glu Ala Glu Phe Asn
Tyr Leu 195 200 205Asn Thr Ala Arg
Thr Leu Glu Leu Tyr Gly Val Glu Phe His Tyr Ala 210
215 220Arg Asp Gln Ser Asn Asn Glu Ile Met Ile Gly Val
Met Ser Gly Gly225 230 235
240Ile Leu Ile Tyr Lys Asn Arg Val Arg Met Asn Thr Phe Pro Trp Leu
245 250 255Lys Ile Val Lys Ile
Ser Phe Lys Cys Lys Gln Phe Phe Ile Gln Leu 260
265 270Arg Lys Glu Leu His Glu Ser Arg Glu Thr Leu Leu
Gly Phe Asn Met 275 280 285Val Asn
Tyr Arg Ala Cys Lys Asn Leu Trp Lys Ala Cys Val Glu His 290
295 300His Thr Phe Phe Arg Leu Asp Arg Pro Leu Pro
Pro Gln Lys Asn Phe305 310 315
320Phe Ala His Tyr Phe Thr Leu Gly Ser Lys Phe Arg Tyr Cys Gly Arg
325 330 335Thr Glu Val Gln
Ser Val Gln Tyr Gly Lys Glu Lys Ala Asn Lys Asp 340
345 350Arg Val Phe Ala Arg Ser Pro Ser Lys Pro Leu
Ala Arg Lys Leu Met 355 360 365Asp
Trp Glu Val Val Ser Arg Asn Ser Ile Ser Asp Asp Arg Leu Glu 370
375 380Thr Gln Ser Leu Pro Ser Arg Ser Pro Pro
Gly Thr Pro Asn His Arg385 390 395
400Asn Ser Thr Phe Thr Gln Glu Gly Thr Arg Leu Arg Pro Ser Ser
Val 405 410 415Gly His Leu
Val Asp His Met Val His Thr Ser Pro Ser Glu Val Phe 420
425 430Val Asn Gln Arg Ser Pro Ser Ser Thr Gln
Ala Asn Ser Ile Val Leu 435 440
445Glu Ser Ser Pro Ser Gln Glu Thr Pro Gly Asp Gly Lys Pro Pro Ala 450
455 460Leu Pro Pro Lys Gln Ser Lys Lys
Asn Ser Trp Asn Gln Ile His Tyr465 470
475 480Ser His Ser Gln Gln Asp Leu Glu Ser His Ile Asn
Glu Thr Phe Asp 485 490
495Ile Pro Ser Ser Pro Glu Lys Pro Thr Pro Asn Gly Gly Ile Pro His
500 505 510Asp Asn Leu Val Leu Ile
Arg Met Lys Pro Asp Glu Asn Gly Arg Phe 515 520
525Gly Phe Asn Val Lys Gly Gly Tyr Asp Gln Lys Met Pro Val
Ile Val 530 535 540Ser Arg Val Ala Pro
Gly Thr Pro Ala Asp Leu Cys Val Pro Arg Leu545 550
555 560Asn Glu Gly Asp Gln Val Val Leu Ile Asn
Gly Arg Asp Ile Ala Glu 565 570
575His Thr His Asp Gln Val Val Leu Phe Ile Lys Ala Ser Cys Glu Arg
580 585 590His Ser Gly Glu Leu
Met Leu Leu Val Arg Pro Asn Ala Val Tyr Asp 595
600 605Val Val Glu Glu Lys Leu Glu Asn Glu Pro Asp Phe
Gln Tyr Ile Pro 610 615 620Glu Lys Ala
Pro Leu Asp Ser Val His Gln Asp Asp His Ser Leu Arg625
630 635 640Glu Ser Met Ile Gln Leu Ala
Glu Gly Leu Ile Thr Gly Thr Val Leu 645
650 655Thr Gln Phe Asp Gln Leu Tyr Arg Lys Lys Pro Gly
Met Thr Met Ser 660 665 670Cys
Ala Lys Leu Pro Gln Asn Ile Ser Lys Asn Arg Tyr Arg Asp Ile 675
680 685Ser Pro Tyr Asp Ala Thr Arg Val Ile
Leu Lys Gly Asn Glu Asp Tyr 690 695
700Ile Asn Ala Asn Tyr Ile Asn Met Glu Ile Pro Ser Ser Ser Ile Ile705
710 715 720Asn Gln Tyr Ile
Ala Cys Gln Gly Pro Leu Pro His Thr Cys Thr Asp 725
730 735Phe Trp Gln Met Thr Trp Glu Gln Gly Ser
Ser Met Val Val Met Leu 740 745
750Thr Thr Gln Val Glu Arg Gly Arg Val Lys Cys His Gln Tyr Trp Pro
755 760 765Glu Pro Thr Gly Ser Ser Ser
Tyr Gly Cys Tyr Gln Val Thr Cys His 770 775
780Ser Glu Glu Gly Asn Thr Ala Tyr Ile Phe Arg Lys Met Thr Leu
Phe785 790 795 800Asn Gln
Glu Lys Asn Glu Ser Arg Pro Leu Thr Gln Ile Gln Tyr Ile
805 810 815Ala Trp Pro Asp His Gly Val
Pro Asp Asp Ser Ser Asp Phe Leu Asp 820 825
830Phe Val Cys His Val Arg Asn Lys Arg Ala Gly Lys Glu Glu
Pro Val 835 840 845Val Val His Cys
Ser Ala Gly Ile Gly Arg Thr Gly Val Leu Ile Thr 850
855 860Met Glu Thr Ala Met Cys Leu Ile Glu Cys Asn Gln
Pro Val Tyr Pro865 870 875
880Leu Asp Ile Val Arg Thr Met Arg Asp Gln Arg Ala Met Met Ile Gln
885 890 895Thr Pro Ser Gln Tyr
Arg Phe Val Cys Glu Ala Ile Leu Lys Val Tyr 900
905 910Glu Glu Gly Phe Val Lys Pro Leu Thr Thr Ser Thr
Asn Lys 915 920
92510806PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Asp Gln Arg Glu Ile Leu Gln Lys Phe Leu Asp
Glu Ala Gln Ser Lys1 5 10
15Lys Ile Thr Lys Glu Glu Phe Ala Asn Glu Phe Leu Lys Leu Lys Arg
20 25 30Gln Ser Thr Lys Tyr Lys Ala
Asp Lys Thr Tyr Pro Thr Thr Val Ala 35 40
45Glu Lys Pro Lys Asn Ile Lys Lys Asn Arg Tyr Lys Asp Ile Leu
Pro 50 55 60Tyr Asp Tyr Ser Arg Val
Glu Leu Ser Leu Ile Thr Ser Asp Glu Asp65 70
75 80Ser Ser Tyr Ile Asn Ala Asn Phe Ile Lys Gly
Val Tyr Gly Pro Lys 85 90
95Ala Tyr Ile Ala Thr Gln Gly Pro Leu Ser Thr Thr Leu Leu Asp Phe
100 105 110Trp Arg Met Ile Trp Glu
Tyr Ser Val Leu Ile Ile Val Met Ala Cys 115 120
125Met Glu Tyr Glu Met Gly Lys Lys Lys Cys Glu Arg Tyr Trp
Ala Glu 130 135 140Pro Gly Glu Met Gln
Leu Glu Phe Gly Pro Phe Ser Val Ser Cys Glu145 150
155 160Ala Glu Lys Arg Lys Ser Asp Tyr Ile Ile
Arg Thr Leu Lys Val Lys 165 170
175Phe Asn Ser Glu Thr Arg Thr Ile Tyr Gln Phe His Tyr Lys Asn Trp
180 185 190Pro Asp His Asp Val
Pro Ser Ser Ile Asp Pro Ile Leu Glu Leu Ile 195
200 205Trp Asp Val Arg Cys Tyr Gln Glu Asp Asp Ser Val
Pro Ile Cys Ile 210 215 220His Cys Ser
Ala Gly Cys Gly Arg Thr Gly Val Ile Cys Ala Ile Asp225
230 235 240Tyr Thr Trp Met Leu Leu Lys
Asp Gly Ile Ile Pro Glu Asn Phe Ser 245
250 255Val Phe Ser Leu Ile Arg Glu Met Arg Thr Gln Arg
Pro Ser Leu Val 260 265 270Gln
Thr Gln Glu Gln Tyr Glu Leu Val Tyr Asn Ala Val Leu Glu Leu 275
280 285Phe Lys Arg Gln Met Asp Val Ile Arg
Asp Lys His Ser Gly Thr Glu 290 295
300Ser Gln Ala Lys His Cys Ile Pro Glu Lys Asn His Thr Leu Gln Ala305
310 315 320Asp Ser Tyr Ser
Pro Asn Leu Pro Lys Ser Thr Thr Lys Ala Ala Lys 325
330 335Met Met Asn Gln Gln Arg Thr Lys Met Glu
Ile Lys Glu Ser Ser Ser 340 345
350Phe Asp Phe Arg Thr Ser Glu Ile Ser Ala Lys Glu Glu Leu Val Leu
355 360 365His Pro Ala Lys Ser Ser Thr
Ser Phe Asp Phe Leu Glu Leu Asn Tyr 370 375
380Ser Phe Asp Lys Asn Ala Asp Thr Thr Met Lys Trp Gln Thr Lys
Ala385 390 395 400Phe Pro
Ile Val Gly Glu Pro Leu Gln Lys His Gln Ser Leu Asp Leu
405 410 415Gly Ser Leu Leu Phe Glu Gly
Cys Ser Asn Ser Lys Pro Val Asn Ala 420 425
430Ala Gly Arg Tyr Phe Asn Ser Lys Val Pro Ile Thr Arg Thr
Lys Ser 435 440 445Thr Pro Phe Glu
Leu Ile Gln Gln Arg Glu Thr Lys Glu Val Asp Ser 450
455 460Lys Glu Asn Phe Ser Tyr Leu Glu Ser Gln Pro His
Asp Ser Cys Phe465 470 475
480Val Glu Met Gln Ala Gln Lys Val Met His Val Ser Ser Ala Glu Leu
485 490 495Asn Tyr Ser Leu Pro
Tyr Asp Ser Lys His Gln Ile Arg Asn Ala Ser 500
505 510Asn Val Lys His His Asp Ser Ser Ala Leu Gly Val
Tyr Ser Tyr Ile 515 520 525Pro Leu
Val Glu Asn Pro Tyr Phe Ser Ser Trp Pro Pro Ser Gly Thr 530
535 540Ser Ser Lys Met Ser Leu Asp Leu Pro Glu Lys
Gln Asp Gly Thr Val545 550 555
560Phe Pro Ser Ser Leu Leu Pro Thr Ser Ser Thr Ser Leu Phe Ser Tyr
565 570 575Tyr Asn Ser His
Asp Ser Leu Ser Leu Asn Ser Pro Thr Asn Ile Ser 580
585 590Ser Leu Leu Asn Gln Glu Ser Ala Val Leu Ala
Thr Ala Pro Arg Ile 595 600 605Asp
Asp Glu Ile Pro Pro Pro Leu Pro Val Arg Thr Pro Glu Ser Phe 610
615 620Ile Val Val Glu Glu Ala Gly Glu Phe Ser
Pro Asn Val Pro Lys Ser625 630 635
640Leu Ser Ser Ala Val Lys Val Lys Ile Gly Thr Ser Leu Glu Trp
Gly 645 650 655Gly Thr Ser
Glu Pro Lys Lys Phe Asp Asp Ser Val Ile Leu Arg Pro 660
665 670Ser Lys Ser Val Lys Leu Arg Ser Pro Lys
Ser Glu Leu His Gln Asp 675 680
685Arg Ser Ser Pro Pro Pro Pro Leu Pro Glu Arg Thr Leu Glu Ser Phe 690
695 700Phe Leu Ala Asp Glu Asp Cys Met
Gln Ala Gln Ser Ile Glu Thr Tyr705 710
715 720Ser Thr Ser Tyr Pro Asp Thr Met Glu Asn Ser Thr
Ser Ser Lys Gln 725 730
735Thr Leu Lys Thr Pro Gly Lys Ser Phe Thr Arg Ser Lys Ser Leu Lys
740 745 750Ile Leu Arg Asn Met Lys
Lys Ser Ile Cys Asn Ser Cys Pro Pro Asn 755 760
765Lys Pro Ala Glu Ser Val Gln Ser Asn Asn Ser Ser Ser Phe
Leu Asn 770 775 780Phe Gly Phe Ala Asn
Arg Phe Ser Lys Pro Lys Gly Pro Arg Asn Pro785 790
795 800Pro Pro Thr Trp Asn Ile
80511254PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe
Tyr Gly Ser Ile Ser1 5 10
15Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly
20 25 30Leu Phe Leu Leu Arg Gln Cys
Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40
45Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg
Gln 50 55 60Leu Asn Gly Thr Tyr Ala
Ile Ala Gly Gly Lys Ala His Cys Gly Pro65 70
75 80Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro
Asp Gly Leu Pro Cys 85 90
95Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro
100 105 110Gly Val Phe Asp Cys Leu
Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120
125Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile
Ile Ser 130 135 140Gln Ala Pro Gln Val
Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg145 150
155 160Met Pro Trp Tyr His Ser Ser Leu Thr Arg
Glu Glu Ala Glu Arg Lys 165 170
175Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg
180 185 190Lys Glu Gln Gly Thr
Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195
200 205Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys
Tyr Cys Ile Pro 210 215 220Glu Gly Thr
Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys225
230 235 240Leu Lys Ala Asp Gly Leu Ile
Tyr Cys Leu Lys Glu Ala Cys 245
25012337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe
Tyr Gly Ser Ile Ser1 5 10
15Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly
20 25 30Leu Phe Leu Leu Arg Gln Cys
Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40
45Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg
Gln 50 55 60Leu Asn Gly Thr Tyr Ala
Ile Ala Gly Gly Lys Ala His Cys Gly Pro65 70
75 80Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro
Asp Gly Leu Pro Cys 85 90
95Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro
100 105 110Gly Val Phe Asp Cys Leu
Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120
125Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile
Ile Ser 130 135 140Gln Ala Pro Gln Val
Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg145 150
155 160Met Pro Trp Tyr His Ser Ser Leu Thr Arg
Glu Glu Ala Glu Arg Lys 165 170
175Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg
180 185 190Lys Glu Gln Gly Thr
Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195
200 205Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys
Tyr Cys Ile Pro 210 215 220Glu Gly Thr
Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys225
230 235 240Leu Lys Ala Asp Gly Leu Ile
Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245
250 255Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro
Thr Leu Pro Ala 260 265 270His
Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn 275
280 285Ser Asp Gly Tyr Thr Pro Glu Pro Ala
Arg Ile Thr Ser Pro Asp Lys 290 295
300Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser305
310 315 320Asp Pro Glu Glu
Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 325
330 335Leu13619PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 13Met Pro Asp Pro Ala
Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser1 5
10 15Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala
Gly Met Ala Asp Gly 20 25
30Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu
35 40 45Ser Leu Val His Asp Val Arg Phe
His His Phe Pro Ile Glu Arg Gln 50 55
60Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro65
70 75 80Ala Glu Leu Cys Glu
Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85
90 95Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly
Leu Glu Pro Gln Pro 100 105
110Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg
115 120 125Gln Thr Trp Lys Leu Glu Gly
Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135
140Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu
Arg145 150 155 160Met Pro
Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys
165 170 175Leu Tyr Ser Gly Ala Gln Thr
Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185
190Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys
Thr Val 195 200 205Tyr His Tyr Leu
Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210
215 220Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val
Glu Tyr Leu Lys225 230 235
240Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn
245 250 255Ser Ser Ala Ser Asn
Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260
265 270His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile
Asp Thr Leu Asn 275 280 285Ser Asp
Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys 290
295 300Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr
Glu Ser Pro Tyr Ser305 310 315
320Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn
325 330 335Leu Leu Ile Ala
Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 340
345 350Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln
Ile Asp Val Ala Ile 355 360 365Lys
Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 370
375 380Arg Glu Ala Gln Ile Met His Gln Leu Asp
Asn Pro Tyr Ile Val Arg385 390 395
400Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu
Met 405 410 415Ala Gly Gly
Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 420
425 430Ile Pro Val Ser Asn Val Ala Glu Leu Leu
His Gln Val Ser Met Gly 435 440
445Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 450
455 460Arg Asn Val Leu Leu Val Asn Arg
His Tyr Ala Lys Ile Ser Asp Phe465 470
475 480Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Phe
Phe Thr Ala Arg 485 490
495Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn
500 505 510Phe Arg Lys Phe Ser Ser
Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 515 520
525Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys
Met Lys 530 535 540Gly Pro Glu Val Met
Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys545 550
555 560Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala
Leu Met Ser Asp Cys Trp 565 570
575Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg
580 585 590Met Arg Ala Cys Tyr
Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 595
600 605Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 610
61514317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Asp Met Ile Thr Ile Phe Ile Gly Thr
Trp Asn Met Gly Asn Ala Pro1 5 10
15Pro Pro Lys Lys Ile Thr Ser Trp Phe Leu Ser Lys Gly Gln Gly
Lys 20 25 30Thr Arg Asp Asp
Ser Ala Asp Tyr Ile Pro His Asp Ile Tyr Val Ile 35
40 45Gly Thr Gln Glu Asp Pro Leu Ser Glu Lys Glu Trp
Leu Glu Ile Leu 50 55 60Lys His Ser
Leu Gln Glu Ile Thr Ser Val Thr Phe Lys Thr Val Ala65 70
75 80Ile His Thr Leu Trp Asn Ile Arg
Ile Val Val Leu Ala Lys Pro Glu 85 90
95His Glu Asn Arg Ile Ser His Ile Cys Thr Asp Asn Val Lys
Thr Gly 100 105 110Ile Ala Asn
Thr Leu Gly Asn Lys Gly Ala Val Gly Val Ser Phe Met 115
120 125Phe Asn Gly Thr Ser Leu Gly Phe Val Asn Ser
His Leu Thr Ser Gly 130 135 140Ser Glu
Lys Lys Leu Arg Arg Asn Gln Asn Tyr Met Asn Ile Leu Arg145
150 155 160Phe Leu Ala Leu Gly Asp Lys
Lys Leu Ser Pro Phe Asn Ile Thr His 165
170 175Arg Phe Thr His Leu Phe Trp Phe Gly Asp Leu Asn
Tyr Arg Val Asp 180 185 190Leu
Pro Thr Trp Glu Ala Glu Thr Ile Ile Gln Lys Ile Lys Gln Gln 195
200 205Gln Tyr Ala Asp Leu Leu Ser His Asp
Gln Leu Leu Thr Glu Arg Arg 210 215
220Glu Gln Lys Val Phe Leu His Phe Glu Glu Glu Glu Ile Thr Phe Ala225
230 235 240Pro Thr Tyr Arg
Phe Glu Arg Leu Thr Arg Asp Lys Tyr Ala Tyr Thr 245
250 255Lys Gln Lys Ala Thr Gly Met Lys Tyr Asn
Leu Pro Ser Trp Cys Asp 260 265
270Arg Val Leu Trp Lys Ser Tyr Pro Leu Val His Val Val Cys Gln Ser
275 280 285Tyr Gly Ser Thr Ser Asp Ile
Met Thr Ser Asp His Ser Pro Val Phe 290 295
300Ala Thr Phe Glu Ala Gly Val Thr Ser Gln Phe Val Ser305
310 315151347DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 15agtgccattc aggctgcatg
gccgtcaggt acggagtgta ttgctaaata caattttcac 60gggactgctg aacaggacct
tcctttttgc aaaggggacg tcttgactat cgtagctgtg 120acgaaagacc cgaactggta
caaagctaag aataaggtcg gccgggaggg tataattccc 180gcaaattacg tgcagaagcg
agaaggtgtt aaagctggaa caaagttgtc actcatgccg 240tggtttcatg gaaaaattac
cagagaacaa gcggagcggc tgttgtaccc gccggaaacc 300ggccttttct tggttaggga
aagcaccaat taccctgggg actacactct ttgtgtttca 360tgcgatggga aggtagaaca
ttaccgcatc atgtatcatg ccagtaagct ttccatagac 420gaagaggtgt acttcgagaa
cctgatgcaa ctggtggagc actacacatc cgatgcggac 480ggattgtgca cgcgattgat
aaaaccaaag gtaatggaag gcaccgtggc agctcaggat 540gagttttacc gaagcggttg
ggcactgaac atgaaagaac ttaaactgct tcagacaatt 600gggaaaggag aatttggcga
cgtgatgctg ggtgattata ggggtaacaa ggtcgcggtg 660aagtgcatta agaacgatgc
cacggcccag gcctttctgg cagaagcttc agttatgact 720caactccgcc attctaatct
ggtccaattg cttggagtga tcgtcgaaga gaagggaggc 780ctctacatag taacggagta
tatggctaaa ggttctctcg tcgattatct ccgctcacgc 840gggcgcagcg ttctcggggg
agattgcctc ctgaaattca gtctggacgt ctgcgaggct 900atggagtacc tcgaaggaaa
caattttgtc caccgggact tggctgcaag gaacgtcctg 960gtaagcgaag ataacgtggc
taaggtgtcc gacttcgggc tcacgaaaga agcaagtagt 1020actcaagaca cgggcaaact
cccagttaaa tggaccgcac cggaggcact cagggaaaaa 1080aaattttcta ccaagagtga
cgtatggtca ttcggcatcc tcttgtggga aatatatagt 1140tttgggcgcg ttccttaccc
aagaatcccc ctgaaggatg tcgtgccgag agtggaaaag 1200gggtacaaga tggatgctcc
ggacggatgc cccccagcag tatatgaggt aatgaaaaat 1260tgctggcatc tcgatgcagc
gatgcggccg tcctttcttc agctccggga gcaactggaa 1320cacataaaga cgcacgaact
tcatctt 1347161140DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
16gttaaagctg gaacaaagtt gtcactcatg ccgtggtttc atggaaaaat taccagagaa
60caagcggagc ggctgttgta cccgccggaa accggccttt tcttggttag ggaaagcacc
120aattaccctg gggactacac tctttgtgtt tcatgcgatg ggaaggtaga acattaccgc
180atcatgtatc atgccagtaa gctttccata gacgaagagg tgtacttcga gaacctgatg
240caactggtgg agcactacac atccgatgcg gacggattgt gcacgcgatt gataaaacca
300aaggtaatgg aaggcaccgt ggcagctcag gatgagtttt accgaagcgg ttgggcactg
360aacatgaaag aacttaaact gcttcagaca attgggaaag gagaatttgg cgacgtgatg
420ctgggtgatt ataggggtaa caaggtcgcg gtgaagtgca ttaagaacga tgccacggcc
480caggcctttc tggcagaagc ttcagttatg actcaactcc gccattctaa tctggtccaa
540ttgcttggag tgatcgtcga agagaaggga ggcctctaca tagtaacgga gtatatggct
600aaaggttctc tcgtcgatta tctccgctca cgcgggcgca gcgttctcgg gggagattgc
660ctcctgaaat tcagtctgga cgtctgcgag gctatggagt acctcgaagg aaacaatttt
720gtccaccggg acttggctgc aaggaacgtc ctggtaagcg aagataacgt ggctaaggtg
780tccgacttcg ggctcacgaa agaagcaagt agtactcaag acacgggcaa actcccagtt
840aaatggaccg caccggaggc actcagggaa aaaaaatttt ctaccaagag tgacgtatgg
900tcattcggca tcctcttgtg ggaaatatat agttttgggc gcgttcctta cccaagaatc
960cccctgaagg atgtcgtgcc gagagtggaa aaggggtaca agatggatgc tccggacgga
1020tgccccccag cagtatatga ggtaatgaaa aattgctggc atctcgatgc agcgatgcgg
1080ccgtcctttc ttcagctccg ggagcaactg gaacacataa agacgcacga acttcatctt
1140171785DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 17atggttcgat ggttccacag agatctgagc
ggcctggatg ccgagactct gcttaaagga 60cggggcgtgc acggcagctt tctggctaga
cccagcagaa agaaccaggg cgacttcagc 120ctgtccgtca gagtgggcga tcaagtgaca
cacatcagaa tccagaactc cggcgacttc 180tacgacctgt acggcggcga gaagttcgcc
acactgacag agctggtgga atattacacc 240cagcagcaag gcgtgctgca ggacagagat
ggcaccatca tccacctgaa gtaccctctg 300aactgcagcg accccaccag cgagagatgg
tatcacggac acatgtctgg cggccaggct 360gagacactgc ttcaggctaa aggcgagccc
tggacctttc tcgtgcggga atctctgagt 420cagcccggcg attttgtgct gagcgtgctg
tccgatcagc ccaaagctgg accaggctct 480ccactgagag tgacccatat caaagtgatg
tgcgaaggcg gacggtacac cgtcggtggc 540ctggaaacat tcgatagcct gaccgacctg
gtcgagcact tcaagaaaac cggcatcgag 600gaagccagcg gcgccttcgt ttatctgaga
cagccctact acgccacaag agtgaacgcc 660gccgacatcg agaacagagt gctggaactg
aacaagaagc aagagagcga ggataccgcc 720aaggccggct tctgggaaga gttcgagtcc
ctgcagaaac aagaagtgaa gaacctgcac 780cagcggctgg aaggacagag gcctgagaac
aagggcaaga accggtacaa gaacatcctg 840ccattcgacc actccagagt gatcctgcag
ggaagagaca gcaacatccc cggctccgac 900tacatcaacg ccaattacat caagaaccag
ctgctgggcc ccgacgagaa cgccaagaca 960tatatcgcct ctcagggctg cctggaagcc
accgtgaacg acttttggca gatggcctgg 1020caagagaaca gccgcgtgat cgtgatgacc
accagagagg tggaaaaagg ccggaacaaa 1080tgcgtgccct actggcccga agtgggcatg
caaagagcct acggacctta cagcgtgacc 1140aactgcggcg agcacgatac caccgagtac
aagctgagaa ccctccaggt gtcccctctg 1200gacaacggcg acctgatcag agagatctgg
cactaccagt acctgtcctg gcctgatcac 1260ggcgtgccat ctgaacctgg tggcgtgctg
agtttcctgg accagatcaa ccagagacaa 1320gagagcctgc ctcacgctgg ccctatcatc
gtgcactgtt ctgccggcat cggcagaacc 1380ggcacaatca tcgtgatcga catgctgatg
gaaaacatca gcaccaaggg cctcgactgc 1440gacatcgaca tccagaaaac catccagatg
gttcgagccc agcggagcgg aatggtgcag 1500acagaagccc agtacaagtt catctacgtg
gcaatcgccc agttcatcga aaccaccaag 1560aaaaagctgg aagtgctgca gtcccagaag
ggccaagaaa gcgagtacgg caacatcaca 1620taccctccag ccatgaagaa cgcccacgcc
aaagccagca gaacctccag caagcacaaa 1680gaggacgtct acgagaatct gcacacaaag
aacaagcgcg aggaaaaagt gaaaaagcag 1740cgcagcgccg acaaagagaa gtccaagggc
agcctgaaga gaaag 178518816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
18ttctgggaag agttcgagtc cctgcagaaa caagaagtga agaacctgca ccagcggctg
60gaaggacaga ggcctgagaa caagggcaag aaccggtaca agaacatcct gccattcgac
120cactccagag tgatcctgca gggaagagac agcaacatcc ccggctccga ctacatcaac
180gccaattaca tcaagaacca gctgctgggc cccgacgaga acgccaagac atatatcgcc
240tctcagggct gcctggaagc caccgtgaac gacttttggc agatggcctg gcaagagaac
300agccgcgtga tcgtgatgac caccagagag gtggaaaaag gccggaacaa atgcgtgccc
360tactggcccg aagtgggcat gcaaagagcc tacggacctt acagcgtgac caactgcggc
420gagcacgata ccaccgagta caagctgaga accctccagg tgtcccctct ggacaacggc
480gacctgatca gagagatctg gcactaccag tacctgtcct ggcctgatca cggcgtgcca
540tctgaacctg gtggcgtgct gagtttcctg gaccagatca accagagaca agagagcctg
600cctcacgctg gccctatcat cgtgcactgt tctgccggca tcggcagaac cggcacaatc
660atcgtgatcg acatgctgat ggaaaacatc agcaccaagg gcctcgactg cgacatcgac
720atccagaaaa ccatccagat ggttcgagcc cagcggagcg gaatggtgca gacagaagcc
780cagtacaagt tcatctacgt ggcaatcgcc cagttc
816191791DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 19atgacaagca gacggtggtt tcaccccaac
atcaccggcg tggaagctga gaatctgctg 60ctgacaagag gcgtggacgg cagctttctg
gctagaccca gcaagtccaa tcctggcgac 120ttcacactga gcgtgcggag aaatggcgcc
gtgacacaca tcaagatcca gaacaccggc 180gactactacg acctgtacgg cggcgagaag
tttgccacac tggcagagct ggtgcagtac 240tacatggaac accacggcca gctgaaagaa
aagaacggcg acgtgatcga gctgaagtac 300cctctgaact gcgccgatcc taccagcgag
agatggtttc acggccacct gtctggcaaa 360gaggccgaga agctgctgac cgagaagggc
aagcacggaa gctttctcgt gcgcgagtct 420cagtctcacc ccggcgattt tgtgctgtct
gtgcggacag gggacgacaa gggcgagagc 480aatgacggca agagcaaagt gacccacgtg
atgatccggt gccaagagct gaaatacgac 540gtcggcggag gggagagatt cgactctctg
accgatctgg tggaacacta caagaaaaac 600cccatggtgg aaaccctggg caccgtgctg
cagctgaagc agccactgaa caccaccaga 660atcaacgccg ccgagatcga gagcagagtg
cgggaactgt ctaagctggc cgagactacc 720gacaaagtga agcaaggctt ctgggaagag
ttcgagacac tgcagcagca agagtgcaag 780ctgctgtact cccggaaaga gggccagaga
caagagaaca agaacaaaaa ccggtacaag 840aacatcctgc cgttcgatca caccagagtg
gtgctgcacg acggcgatcc taatgagccc 900gtgtccgact acatcaacgc caacatcatc
atgcccgagt ttgagacaaa gtgcaacaat 960agcaagccca agaagtccta tatcgccaca
cagggctgcc tgcagaatac cgtgaacgac 1020ttttggcgga tggtgtttca agagaactcc
cgcgtgatcg tgatgaccac caaagaggtg 1080gaacggggca agtctaagtg cgtgaagtac
tggcccgacg agtacgccct gaaagaatac 1140ggcgtgatga gagtgcggaa cgtgaaagag
agcgccgctc acgattacac cctgagagag 1200ctgaagctga gcaaagtcgg acaggccctg
ctgcagggaa acaccgaaag aaccgtgtgg 1260cagtaccact tccggacctg gccagatcat
ggcgtgccat ctgatcctgg cggcgtgctg 1320gatttcctgg aagaggtgca ccacaagcaa
gagtccatca tggacgccgg accagtggtg 1380gtgcactgtt ctgccggaat cggaagaacc
ggcaccttca tcgtgatcga catcctgatt 1440gacatcatcc gcgagaaagg cgtcgactgc
gatatcgacg tgcccaagac catccagatg 1500gttcgaagcc agagaagcgg catggtgcag
acagaggccc agtaccggtt catctacatg 1560gccgtgcagc attacatcga aaccctgcag
cggcggatcg aggaagaaca gaagtccaag 1620agaaagggcc acgagtacac caacatcaag
tacagcctgg ccgaccagac cagcggcgat 1680caatctcctc tgcctccttg cacacccaca
cctccatgtg ccgagatgcg ggaagatagc 1740gccagggtgt acgagaacgt gggcctgatg
caacagcaga agtccttccg g 1791202124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
20tacaagatct acgacctgca caagaagcgg agctgcaatc tggacgagca gcaagaactg
60gtggaacggg acgacgagaa gcagctgatg aacgtggaac ccatccacgc cgacatcctg
120ctggaaacct acaagcggaa gatcgccgac gagggcagac tgttcctggc cgagtttcag
180agcatcccca gagtgttcag caagttcccc atcaaagagg ccagaaagcc cttcaaccag
240aacaagaacc gctacgtgga cattctgccc tacgactaca atcgcgtgga actgagcgag
300atcaatggcg acgccggcag caactacatc aacgccagct acatcgacgg cttcaaagag
360ccccggaagt atatcgccgc tcagggccct agagatgaga cagtggacga cttctggcgc
420atgatttggg agcagaaagc caccgtgatc gtgatggtta ccagatgcga agagggcaac
480agaaacaagt gcgccgagta ctggcccagc atggaagaag gcacaagagc ctttggcgac
540gtggtggtca agatcaatca gcacaagcgg tgccccgact acatcatcca gaaactgaac
600atcgtgaaca agaaagagaa ggccaccgga cgggaagtga cccacatcca gtttaccagc
660tggcccgatc atggcgtgcc cgaggatcca catctgctgc tcaagctgcg gagaagagtg
720aacgccttca gcaacttctt cagcggcccc atcgtggtgc actgttctgc aggcgttgga
780agaaccggca cctacatcgg aatcgacgcc atgctggaag gactggaagc cgagaacaag
840gtggacgtgt acggctacgt ggtcaagctg agaaggcagc ggtgtctgat ggtgcaggtt
900gaggcccagt acatcctgat ccatcaggcc ctggtcgagt acaaccagtt cggcgagaca
960gaagtgaacc tgagcgagct gcacccctat ctgcacaaca tgaagaagcg ggaccctcca
1020agcgagccct ctccactgga agctgagttc cagagactgc ccagctacag aagctggcgg
1080acacagcaca tcggcaatca agaggaaaac aagagcaaga accggaacag caacgtgatc
1140ccgtacgatt acaacagagt gcccctgaag cacgaactcg agatgagcaa agagagcgag
1200cacgacagcg acgagtccag cgacgatgat agcgacagcg aggaacccag caagtatatc
1260aatgcctcct tcatcatgag ctattggaag cccgaagtga tgattgccgc acagggaccc
1320ctgaaagaga caatcggcga cttttggcag atgatcttcc agcggaaagt gaaagtgatc
1380gtcatgctga ccgagctgaa acacggcgac caagagatct gcgcccagta ttggggagag
1440ggaaagcaga cctacggcga cattgaggtg gacctgaagg acaccgacaa gagcagcacc
1500tacacactgc gggtgttcga gctgagacac tccaagagaa aggacagccg gaccgtgtac
1560cagtaccagt ataccaattg gagcgtggaa cagctgcctg ccgagcctaa agaactgatc
1620agcatgatcc aggtcgtgaa gcagaagctg cctcagaaga acagcagcga gggaaacaag
1680caccacaagt ctacccctct gctgatccac tgcagagatg gctctcagca gaccggcatc
1740ttctgcgccc tgctgaatct cctggaaagc gccgagacag aggaagtggt ggacatcttc
1800caggtggtca aagccctgcg gaaggccaga cctggcatgg tgtctacctt cgagcagtat
1860cagttcctgt acgacgtgat cgccagcaca taccccgctc agaacggcca agtgaagaag
1920aacaaccacc aagaggacaa gatcgagttc gacaacgagg tggacaaagt gaagcaggac
1980gccaactgcg tgaaccctct gggagcccca gaaaagctgc ctgaggccaa agaacaggcc
2040gagggctctg agccaacatc tggaacagag ggacctgagc acagcgtgaa cggacctgct
2100agccccgctc tgaatcaggg ctct
212421651DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 21atggaagcca ttgccaaata cgacttcaag
gccaccgccg acgacgagct gagcttcaag 60agaggcgaca tcctgaaggt gctgaacgag
gaatgcgacc agaactggta caaggccgag 120ctgaacggca aggacggctt catccccaag
aactacatcg agatgaagcc ccatccatgg 180ttcttcggca agatccccag agccaaggcc
gaagagatgc tgagcaagca gagacacgac 240ggcgcctttc tgatccggga atctgaatct
gcccctggcg acttcagcct gtccgtgaag 300ttcggcaacg acgtgcagca cttcaaggtc
ctgagagatg gcgccggaaa gtacttcctg 360tgggtcgtga agtttaacag cctgaacgag
ctggtggact accacagatc taccagcgtg 420tcccggaacc agcagatctt cctgcgggac
atcgaacagg ttccccagca acctacctac 480gtgcaggccc tgttcgactt cgaccctcaa
gaggatggcg agctgggctt tagacggggc 540gatttcatcc acgtgatgga caatagcgac
cccaactggt ggaagggcgc ctgtcatgga 600cagaccggca tgttccccag aaactacgtg
acccctgtga accggaacgt g 651221209DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
22atgacagcca tcatcaaaga aatcgtgtcc cggaacaagc ggcgctacca agaggatggc
60ttcgacctgg acctgaccta catctacccc aacatcattg ccatgggctt ccccgccgaa
120agactggaag gcgtgtacag aaacaacatc gacgatgtcg tgcggttcct ggacagcaag
180cacaagaacc actacaagat ctacaacctg tgcgccgagc ggcactacga taccgccaag
240ttcaactgca gagtggctca gtaccccttc gaggaccaca atcctccaca gctggaactg
300atcaagccct tctgcgagga cctggatcag tggctgagcg aggacgataa tcacgtggcc
360gccattcact gcaaggccgg caagggaaga accggcgtga tgatctgtgc ctacctgctg
420caccggggca agtttctgaa agcccaagag gccctggact tctacggcga agtgcggacc
480agagacaaga aaggcgtgac aatccccagc cagcggagat acgtgtacta ctacagctat
540ctgctgaaga accacctgga ctacagaccc gtggcactgc tgttccacaa gatgatgttc
600gagactatcc ccatgttcag cggcggcaca tgcaaccctc agttcgtcgt gtgccagctg
660aaagtgaaga tctactccag caacagcggc cccaccagac gcgaggacaa gttcatgtac
720ttcgagttcc ctcagcctct gcctgtgtgc ggcgacatca aggtggaatt cttccacaag
780cagaacaaga tgctgaaaaa ggacaagatg ttccacttct gggtcaacac cttcttcatc
840cccggacctg aagagacaag cgagaaggtg gaaaacggca gcctgtgcga ccaagagatc
900gacagcatct gcagcatcga gcgggccgac aacgacaaag aatacctggt gctgaccctg
960accaagaacg atctggacaa ggccaacaag gataaggcca accggtactt cagccccaac
1020ttcaaagtga aactgtactt caccaagacc gtcgaggaac ccagcaatcc tgaggccagc
1080tctagcacat ctgtgacccc tgacgtgtcc gacaatgagc ccgaccacta cagatacagc
1140gacaccaccg acagcgaccc cgagaacgag cctttcgatg aggatcagca cacacagatc
1200accaaagtg
1209232778DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 23atgacaagca gattcagact gccagccggc
agaacctaca atgtgcgggc ttctgagctg 60gccagagaca gacagcacac cgaggtcgtg
tgcaacatcc tgctgctcga caataccgtg 120caggccttca aagtgaacaa gcacgaccag
ggccaagtcc tgctggacgt ggtgttcaag 180cacctggatc tgaccgagca ggactacttc
ggactgcagc tggccgacga cagcaccgac 240aatcctagat ggctggaccc caacaagccc
atccggaagc agctgaagag aggctcccct 300tactctctga acttccgggt caagttcttc
gtgtccgatc ctaacaaact gcaagaagag 360tatacccgct accagtactt cctgcagatc
aagcaggaca tcctgaccgg cagactgccc 420tgtccttcta atactgccgc tctgctggcc
tcctttgccg tgcaatctga actgggcgac 480tacgaccaga gcgagaacct gagcggctac
ctgagcgatt acagcttcat ccccaaccag 540cctcaggact tcgagaaaga gatcgccaag
ctgcatcagc agcacatcgg actgtctcca 600gccgaggccg agttcaacta cctgaacacc
gccaggacac tggaactgta cggcgtggaa 660tttcactacg cccgggacca gagcaacaac
gagatcatga ttggcgtgat gagcggcggc 720atcctgatct acaagaacag agtgcggatg
aacacattcc cctggctgaa gatcgtgaag 780atcagcttca agtgcaagca gttcttcatc
cagctgcgga aagagctgca cgagagcaga 840gagacactgc tgggcttcaa catggtcaac
taccgggcct gcaagaacct gtggaaggcc 900tgtgtggaac accacacctt tttccggctg
gaccgacctc tgcctcctca gaagaatttc 960ttcgcccact acttcaccct gggcagcaag
ttcagatact gcggcagaac agaggtgcag 1020tccgtgcagt acggcaaaga gaaagccaac
aaggaccggg tgttcgccag atctcctagc 1080aagccactgg ccagaaagct gatggactgg
gaagtcgtgt cccggaacag catcagcgac 1140gacagactgg aaacccagag cctgcctagc
agaagccctc ctggcacacc caaccacaga 1200aacagcacct tcacacaaga gggcacaaga
ctgaggccta gctctgtggg acacctggtg 1260gatcacatgg tgcacacaag ccccagcgag
gtgttcgtga accagagaag ccctagctct 1320acccaggcca acagcatcgt gctggaaagc
agccccagcc aagaaacacc aggcgacgga 1380aaacctcctg ctctgccacc taagcagagc
aagaagaaca gctggaacca gatccactac 1440agccacagcc agcaggatct ggaaagccac
atcaacgaga cattcgacat ccctagcagc 1500cccgagaagc ccacacctaa tggcggaatc
cctcacgaca acctggtgct gatccggatg 1560aagcccgacg agaatggcag attcggcttc
aacgtgaaag gcggctacga tcagaaaatg 1620cccgtgatcg tgtccagagt ggcccctgga
actcctgccg atctgtgtgt gcccagactg 1680aacgaaggcg accaggtcgt gctgatcaac
ggcagagata tcgccgagca cacccacgat 1740caggtggtgc tgttcattaa ggcctcttgc
gagagacaca gcggcgaact gatgctgctc 1800gtgcggccta atgccgtgta cgacgtggtg
gaagagaaac tggaaaacga gcccgacttc 1860cagtacatcc ctgagaaggc cccactggac
agcgtgcacc aggatgatca tagcctgcgc 1920gagagcatga tccagctggc agagggactg
atcaccggca cagtgctgac ccagttcgac 1980cagctgtacc ggaagaaacc tggcatgacc
atgtcctgcg ccaaactgcc tcagaacatc 2040agcaagaacc ggtacagaga catcagcccc
tacgatgcca ccagagtgat cctgaagggc 2100aacgaggact acatcaacgc caattacatc
aacatggaaa tccccagctc cagcatcatc 2160aaccagtata tcgcctgtca gggcccactg
cctcacacct gtaccgactt ttggcagatg 2220acctgggagc agggcagcag catggtggtc
atgctgacaa cccaggtgga acggggcaga 2280gtgaagtgcc accagtattg gcctgagcct
accggcagca gctcctacgg ctgttaccaa 2340gtgacctgcc acagcgaaga gggcaacacc
gcctacatct tcagaaagat gaccctgttc 2400aatcaagaga agaacgagag ccggcctctg
acacagatcc agtatattgc ttggcccgac 2460cacggcgtgc ccgacgatag ttctgacttc
ctggacttcg tgtgccacgt gcgcaacaaa 2520cgcgccggaa aagaggaacc tgtcgtcgtc
cactgtagcg ccggcattgg aagaaccggc 2580gtgctgatta ccatggaaac agccatgtgc
ctgatcgagt gcaatcagcc cgtgtatccc 2640ctggacatcg tgcggaccat gagagatcag
cgggccatga tgatccagac acctagccag 2700tacagattcg tgtgcgaggc cattctgaag
gtgtacgaag agggattcgt gaagcccctg 2760accacctcca ccaacaag
2778242418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
24gatcagagag agatcctgca gaagttcctg gacgaggccc agagcaagaa gatcaccaaa
60gaggaattcg ccaacgagtt cctgaaactg aagcggcaga gcaccaagta caaggccgac
120aagacatacc ccaccaccgt ggccgagaag cccaagaaca tcaagaagaa ccggtacaag
180gacatcctgc catacgacta ctccagagtg gaactgagcc tgatcaccag cgacgaggac
240agcagctaca tcaacgccaa cttcatcaag ggcgtgtacg gccccaaggc ctatatcgca
300acacagggcc ctctgtctac aaccctgctg gacttctggc gcatgatctg ggagtacagc
360gtgctgatca tcgtgatggc ctgcatggaa tacgagatgg gcaagaagaa gtgcgagcgg
420tactgggccg aacctggcga aatgcagctg gaattcggcc ccttttccgt gtcctgcgaa
480gccgagaaga gaaagtccga ctacatcatc aggaccctga aagtgaagtt caacagcgaa
540acccggacca tctaccagtt tcactacaag aactggcccg accacgacgt gccaagcagc
600atcgatccta tcctggaact gatttgggac gtgcggtgct accaagagga cgacagcgtg
660ccaatctgca tccactgttc tgccggctgc ggaagaacag gcgtcatctg cgccatcgac
720tacacctgga tgctgctgaa ggacggcatc atccccgaga acttcagcgt gttcagcctg
780atccgcgaga tgagaaccca gaggcctagc ctggtgcaga cccaagagca gtacgaactg
840gtgtacaacg ccgtgctgga actgttcaag agacagatgg acgtgatccg ggacaagcac
900agcggcacag agtctcaggc caaacactgc atccctgaga agaatcacac cctgcaggcc
960gacagctaca gccccaatct gcctaagagc accaccaagg ccgccaaaat gatgaaccag
1020cagcggacaa agatggaaat caaagagagc agctccttcg acttccggac cagcgagatc
1080agcgccaaag aagaactggt tctgcacccc gccaagtcct ctaccagctt cgactttctc
1140gagctgaact acagcttcga taagaacgcc gacaccacca tgaagtggca gaccaaggcc
1200tttcctatcg tgggcgagcc tctgcagaaa caccagagcc tggatctggg ctccctgctg
1260tttgagggct gcagcaatag caagcccgtg aacgccgctg gccggtactt taatagcaag
1320gtgcccatca ccaggaccaa gagcacccct ttcgagctga tccagcagcg cgagacaaaa
1380gaggtggaca gcaaagagaa cttctcctac ctggaaagcc agcctcacga cagctgcttc
1440gtggaaatgc aggcccagaa agtgatgcac gtgtccagcg ccgagctgaa ttactctctg
1500ccctacgaca gcaagcacca gatccggaac gccagcaacg tgaagcacca cgatagctct
1560gccctgggag tgtacagcta cattcccctg gtggaaaacc cctacttcag ctcctggcca
1620cctagcggca caagcagcaa gatgtctctg gatctgcccg agaagcagga cggcacagtg
1680ttcccatcta gcctgctgcc taccagcagc accagcctgt tcagctacta caatagccac
1740gactctctgt ccctgaacag ccccaccaac atctccagcc tgctgaatca agaaagcgct
1800gtgctggcca ccgctccaag aatcgacgat gagatccctc ctcctctgcc tgtgcggacc
1860cctgagtctt tcatcgtggt ggaagaggcc ggcgagttca gccctaatgt gcccaaatct
1920ctgagcagcg ccgtgaaagt caagatcggc acctctctgg aatggggcgg cacatccgag
1980cctaagaaat tcgacgactc cgtgatcctg aggccaagca agagcgtgaa gctgagaagc
2040cccaagtccg agctgcatca ggacagatct agccctcctc caccactgcc tgagagaacc
2100ctcgagtcat tcttcctggc cgacgaggat tgcatgcagg cacagagcat cgagacatac
2160agcacaagct accccgacac catggaaaac agcacctcca gcaagcagac actgaaaacc
2220ccaggcaaga gcttcacccg gtccaagagc ctgaagatcc tgcggaacat gaagaagtcc
2280atctgcaaca gctgccctcc aaacaagcct gccgagagcg tgcagtccaa caatagcagc
2340agcttcctga acttcggctt tgccaaccgg ttcagcaagc ctaagggccc cagaaatcct
2400cctcctacat ggaacatc
241825762DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 25atgcctgatc ctgctgctca tctgccattc
ttctacggca gcatcagcag agccgaggcc 60gaagaacatc tgaagctggc cggaatggcc
gacggactgt ttctgctcag acagtgcctg 120agaagcctcg gcggctatgt gctgtctctg
gtgcacgatg tgcggttcca tcacttcccc 180atcgagagac agctgaacgg cacctacgct
atcgctggcg gaaaagccca ttgtggacct 240gccgagctgt gcgagttcta cagcagagat
cctgacggcc tgccttgcaa cctgcggaag 300ccttgcaata gacccagcgg cctggaacct
cagcctggcg ttttcgactg cctgagagat 360gccatggtcc gagattacgt gcggcagacc
tggaagctgg aaggcgaagc tctggaacag 420gcaatcatca gccaggctcc tcaggtggaa
aagctgatcg ccacaacagc ccacgagcgg 480atgccttggt atcacagctc cctgaccaga
gaggaagccg agcggaagct gtattctggc 540gcccagaccg atggcaagtt cctgctgagg
cccagaaaag agcagggcac atacgccctg 600agcctgatct acggcaagac cgtgtaccac
tacctgatct cccaggacaa ggccggcaag 660tactgtatcc ctgagggcac caagttcgac
accctgtggc agctggtgga atacctgaag 720ctgaaagccg atggactgat ctactgcctg
aaagaggcct gc 762261011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
26atgcctgatc ctgctgctca tctgccattc ttctacggca gcatcagcag agccgaggcc
60gaagaacatc tgaagctggc cggaatggcc gacggactgt ttctgctcag acagtgcctg
120agaagcctcg gcggctatgt gctgtctctg gtgcacgatg tgcggttcca tcacttcccc
180atcgagagac agctgaacgg cacctacgct atcgctggcg gaaaagccca ttgtggacct
240gccgagctgt gcgagttcta cagcagagat cctgacggcc tgccttgcaa cctgcggaag
300ccttgcaata gacccagcgg cctggaacct cagcctggcg ttttcgactg cctgagagat
360gccatggtcc gagattacgt gcggcagacc tggaagctgg aaggcgaagc tctggaacag
420gcaatcatca gccaggctcc tcaggtggaa aagctgatcg ccacaacagc ccacgagcgg
480atgccttggt atcacagctc cctgaccaga gaggaagccg agcggaagct gtattctggc
540gcccagaccg atggcaagtt cctgctgagg cccagaaaag agcagggcac atacgccctg
600agcctgatct acggcaagac cgtgtaccac tacctgatct cccaggacaa ggccggcaag
660tactgtatcc ctgagggcac caagttcgac accctgtggc agctggtgga atacctgaag
720ctgaaagccg atggactgat ctactgcctg aaagaggcct gtcctaacag cagcgccagc
780aatgctagcg gagctgctgc acctacactg cctgctcacc ctagcacact gacacaccct
840cagcggagaa tcgataccct gaacagcgac ggctacaccc cagaacctgc cagaatcact
900agccccgaca agcccagacc tatgcctatg gacacctccg tgtacgagag cccctacagc
960gatcccgagg aactgaagga caagaagctg ttcctcaagc gggacaacct g
1011271857DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 27atgcctgatc ctgctgctca tctgccattc
ttctacggca gcatcagcag agccgaggcc 60gaagaacatc tgaagctggc cggaatggcc
gacggactgt ttctgctcag acagtgcctg 120agaagcctcg gcggctatgt gctgtctctg
gtgcacgatg tgcggttcca tcacttcccc 180atcgagagac agctgaacgg cacctacgct
atcgctggcg gaaaagccca ttgtggacct 240gccgagctgt gcgagttcta cagcagagat
cctgacggcc tgccttgcaa cctgcggaag 300ccttgcaata gacccagcgg cctggaacct
cagcctggcg ttttcgactg cctgagagat 360gccatggtcc gagattacgt gcggcagacc
tggaagctgg aaggcgaagc tctggaacag 420gcaatcatca gccaggctcc tcaggtggaa
aagctgatcg ccacaacagc ccacgagcgg 480atgccttggt atcacagctc cctgaccaga
gaggaagccg agcggaagct gtattctggc 540gcccagaccg atggcaagtt cctgctgagg
cccagaaaag agcagggcac atacgccctg 600agcctgatct acggcaagac cgtgtaccac
tacctgatct cccaggacaa ggccggcaag 660tactgtatcc ctgagggcac caagttcgac
accctgtggc agctggtgga atacctgaag 720ctgaaagccg atggactgat ctactgcctg
aaagaggcct gtcctaacag cagcgccagc 780aatgctagcg gagctgctgc acctacactg
cctgctcacc ctagcacact gacacaccct 840cagcggagaa tcgataccct gaacagcgac
ggctacaccc cagaacctgc cagaatcact 900agccccgaca agcccagacc tatgcctatg
gacacctccg tgtacgagag cccctacagc 960gatcccgagg aactgaagga caagaagctg
ttcctcaagc gggacaacct gctgattgcc 1020gacatcgaac tcggctgcgg caattttgga
tctgtgcggc agggcgtgta ccggatgcgg 1080aagaaacaga tcgacgtggc catcaaggtg
ctgaagcagg gaaccgagaa ggccgacacc 1140gaggaaatga tgcgggaagc ccagattatg
caccagctgg acaaccccta catcgtgcgg 1200ctgatcggag tgtgtcaagc cgaggctctg
atgctggtca tggaaatggc aggcggaggc 1260cctctgcaca agtttctcgt tggcaagcgg
gaagagatcc ccgtgtctaa tgtggccgag 1320ctgctccacc aagtgtctat gggcatgaag
tacctggaag agaagaactt cgtgcaccgc 1380gacctggccg ccagaaatgt gctgctggtc
aacagacact acgccaagat cagcgacttc 1440ggcctgtcta aagccctggg cgccgacgat
agcttcttca cagctagaag cgccggaaag 1500tggcccctga agtggtacgc ccctgagtgc
atcaacttcc gcaagttcag ctccagatcc 1560gacgtgtggt cttacggcgt gaccatgtgg
gaagccctga gctacggcca gaaaccttac 1620aagaagatga agggccccga agtcatggcc
ttcatcgaac agggcaagag aatggaatgc 1680cctcctgagt gccctccaga gctgtatgcc
ctgatgagcg attgctggat ctacaagtgg 1740gaagatcggc ccgacttcct gaccgtggaa
cagagaatgc gggcctgcta ctactccctg 1800gcctccaaag ttgagggacc tcctggcagc
acacagaaag ccgaagctgc ttgtgct 185728951DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28gatatgatca ccatcttcat cggcacctgg aatatgggca acgcccctcc acctaagaag
60atcacaagct ggtttctgag caaaggccag ggaaagacca gggacgacag cgccgattac
120atccctcacg atatctacgt gatcgggacc caagaggacc ctctgagcga gaaagagtgg
180ctggaaattc tgaagcactc cctgcaagag atcacctccg tgaccttcaa gaccgtggcc
240atccacacac tgtggaacat ccggatcgtg gtgctggcca agcctgagca cgagaacaga
300atcagccaca tctgcaccga caacgtgaaa accggaatcg ccaacacact gggcaacaaa
360ggcgccgtgg gagtgtcctt catgttcaac ggcaccagcc tgggcttcgt gaacagccac
420ctgacatccg gctccgagaa gaagctgcgg cggaaccaga actatatgaa catcctgcgg
480tttctggccc tgggcgacaa gaagctgagc cccttcaaca tcacccaccg gttcacccac
540ctgttttggt tcggcgacct gaactacaga gtggacctgc ctacctggga agccgagaca
600atcatccaga agatcaagca gcaacagtac gccgacctgc tgtcccacga tcagctgctg
660accgagagaa gggaacagaa agtgttcctg cacttcgagg aagaggaaat cacattcgcc
720cctacctaca gattcgagcg gctgacccgg gataagtacg cctacaccaa gcagaaagcc
780accggcatga agtacaacct gccttcttgg tgcgaccggg tgctgtggaa gtcttaccct
840ctggtgcacg tcgtgtgcca gtcttacggc agcaccagcg acatcatgac cagcgatcac
900agccctgtgt tcgccacatt tgaggccggc gtgaccagcc agtttgtgtc c
951293PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 29Gly Gly Ser1306PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Gly Gly Ser Gly Gly Ser1
5319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gly Gly Ser Gly Gly Ser Gly Gly Ser1
53212PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 32Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser1
5 103315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser1 5 10
15344PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 34Gly Gly Gly Ser1358PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Gly
Gly Gly Ser Gly Gly Gly Ser1 53612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Gly
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5
103716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 37Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser1 5 10
153820PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 38Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
Ser1 5 10 15Gly Gly Gly
Ser 20395PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 39Gly Gly Gly Gly Ser1
54010PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 40Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
104115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 41Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser1 5 10
154220PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 42Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly1 5 10 15Gly Gly
Gly Ser 204325PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 43Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly1 5 10
15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
254418PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 44Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
Thr1 5 10 15Lys
Gly4520PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 45Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala
Lys Glu1 5 10 15Ala Ala
Ala Lys 204642PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 46Ala Ala Ala Ile Glu Val Met Tyr Pro
Pro Pro Tyr Leu Asp Asn Glu1 5 10
15Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys
Pro 20 25 30Ser Pro Leu Phe
Pro Gly Pro Ser Lys Pro 35 404712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro1 5
104812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Glu Ser Lys Tyr Gly Pro Pro Ala Pro Ser Ala Pro1
5 104912PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 49Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro1 5
105012PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 50Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro1
5 105186PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 51Ala Ala Ala Phe Val Pro
Val Phe Leu Pro Ala Lys Pro Thr Thr Thr1 5
10 15Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
Ala Ser Gln Pro 20 25 30Leu
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 35
40 45His Thr Arg Gly Leu Asp Phe Ala Cys
Asp Ile Tyr Ile Trp Ala Pro 50 55
60Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu65
70 75 80Tyr Cys Asn His Arg
Asn 855245PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 52Thr Thr Thr Pro Ala Pro Arg Pro Pro
Thr Pro Ala Pro Thr Ile Ala1 5 10
15Leu Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
Gly 20 25 30Gly Ala Val His
Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40
4553132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 53Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly
Glu Cys Cys Lys Ala1 5 10
15Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr
20 25 30Val Cys Glu Pro Cys Leu Asp
Ser Val Thr Phe Ser Asp Val Val Ser 35 40
45Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln
Ser 50 55 60Met Ser Ala Pro Cys Val
Glu Ala Asp Asp Ala Val Cys Arg Cys Ala65 70
75 80Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg
Cys Glu Ala Cys Arg 85 90
95Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln
100 105 110Asn Thr Val Cys Glu Glu
Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala 115 120
125Asp Ala Glu Cys 1305434PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala1
5 10 15Cys Asn Leu Gly Glu Gly
Val Ala Gln Pro Cys Gly Ala Asn Gln Thr 20 25
30Val Cys5520PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Ala Val Gly Gln Asp Thr Gln
Glu Val Ile Val Val Pro His Ser Leu1 5 10
15Pro Phe Lys Val 2056126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
56gcagcagcta tcgaggtgat gtatcctccg ccctacctgg ataatgaaaa gagtaatggg
60actatcattc atgtaaaagg gaagcatctt tgtccttctc cccttttccc cggtccgtct
120aaacct
1265736DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 57gaaagcaagt acggtccacc ttgccctagc tgtccg
365836DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 58gaatccaagt acggcccccc
agcgcctagt gcccca 365936DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
59gaatctaaat atggcccgcc atgcccgcct tgccca
366036DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 60gaaccgaagt cttgtgataa aactcatacg tgcccg
3661258DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 61gctgctgctt tcgtacccgt gttcctccct
gctaagccta cgactacccc cgcaccgaga 60ccacccacgc cagcacccac gattgctagc
cagcccctta gtttgcgacc agaagcttgt 120cggcctgctg ctggtggcgc ggtacatacc
cgcggccttg attttgcttg cgatatatat 180atctgggcgc ctctggccgg aacatgcggg
gtcctcctcc tttctctggt tattactctc 240tactgtaatc acaggaat
25862396DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
62gcctgcccga ccgggctcta cactcatagc ggggaatgtt gtaaggcatg taacttgggt
60gagggcgtcg cacagccctg cggagctaac caaacagtgt gcgaaccctg cctcgatagt
120gtgacgttct ctgatgttgt atcagctaca gagccttgca aaccatgtac tgagtgcgtt
180ggacttcagt caatgagcgc tccatgtgtg gaggcagatg atgcggtctg tcgatgtgct
240tacggatact accaagacga gacaacaggg cggtgcgagg cctgtagagt ttgtgaggcg
300ggctccgggc tggtgttttc atgtcaagac aagcaaaata cggtctgtga agagtgccct
360gatggcacct actcagacga agcagatgca gaatgc
39663102DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 63gcctgcccta caggactcta cacgcatagc
ggtgagtgtt gtaaagcatg caacctcggg 60gaaggtgtag cccagccatg cggggctaac
caaaccgttt gc 1026460DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
64gctgtgggcc aggacacgca ggaggtcatc gtggtgccac actccttgcc ctttaaggtg
606527PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 65Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
Leu1 5 10 15Leu Val Thr
Val Ala Phe Ile Ile Phe Trp Val 20
256646PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 66Gly Pro Ala Gly Ser Leu Leu Gly Ser Gly Gln Met Gln Ile
Thr Leu1 5 10 15Trp Gly
Ser Leu Ala Ala Val Ala Ile Phe Phe Val Ile Thr Phe Leu 20
25 30Ile Phe Leu Cys Ser Ser Cys Asp Arg
Glu Lys Lys Pro Arg 35 40
456736PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 67Met Glu Ala Asp Ala Leu Ser Pro Val Gly Leu Gly Leu Leu
Leu Leu1 5 10 15Pro Phe
Leu Val Thr Leu Leu Ala Ala Leu Cys Val Arg Cys Arg Glu 20
25 30Leu Pro Val Ser
356836PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 68Met Glu Ala Asp Ala Leu Ser Pro Val Gly Leu Gly Leu Leu
Leu Leu1 5 10 15Pro Phe
Leu Val Thr Leu Leu Ala Ala Leu Ala Val Arg Ala Arg Glu 20
25 30Leu Pro Val Ser
356932PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 69Glu Glu Ala Ile Leu Val Pro Cys Val Leu Gly Leu Leu Leu
Leu Pro1 5 10 15Ile Leu
Ala Met Leu Met Ala Leu Cys Val His Cys His Arg Leu Pro 20
25 307021PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 70Ile Phe Ser Gly Phe Ala
Gly Leu Leu Ala Ile Leu Leu Val Val Ala1 5
10 15Val Phe Cys Ile Leu 207121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Leu
Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu1
5 10 15Thr Ala Leu Phe Leu
207221PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 72Ala Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile
Ala1 5 10 15Leu Leu Val
Val Leu 207381DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 73ttttgggtgc tggtggtggt
tggtggagtc ctggcttgct atagcttgct agtaacagtg 60gcctttatta ttttctgggt g
8174138DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
74ggtccggctg gctctctgct cggcagtggt cagatgcaga ttacgttgtg gggcagtttg
60gcagccgtcg caatcttctt tgttatcact tttcttatct ttctctgttc ctcatgtgac
120agagagaaaa agccccga
13875108DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 75atggaagccg atgctctgtc tcctgttggc
ctgggactgc tgctcctgcc ttttctggtt 60acactgctgg ccgctctgtg tgtgcggtgt
agagaactgc cagttagt 10876108DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
76atggaagccg atgctctgtc tcctgttggc ctgggactgc tgctcctgcc ttttctcgtt
60acactgctgg ccgctctggc tgtgcgagct agagaactgc ctgtgtct
1087796DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77gaggaagcaa tcctggtgcc gtgtgtactt ggtctgcttt
tgttgccaat acttgcgatg 60ctcatggctc tctgcgtaca ttgccatcgg cttccg
967863DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 78atcttcagcg
gctttgccgg actgctggct atcctgctgg ttgtggccgt gttctgtatc 60ctt
637963DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79ctgtgctacc tgctggacgg catcctgttt atctacggcg
tgatcctgac agccctgttc 60ctt
638063DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 80gccctgattg
ccttcctggc ctttctgatc atcgtgacca gcattgccct gctggtcgtg 60ctg
638121PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 81Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala
Arg Pro 208224PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 82Met Glu Thr Asp Thr Leu Leu Leu Trp Val
Leu Leu Leu Trp Val Pro1 5 10
15Gly Ser Thr Gly Ala Gly Gly Ser 208322PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Met
Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1
5 10 15Ala Phe Leu Leu Ile Pro
2084242PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln1 5 10
15Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30Gly Val Ser Trp Ile Arg Gln
Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40
45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu
Lys 50 55 60Ser Arg Leu Thr Ile Ile
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70
75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
Ile Tyr Tyr Cys Ala 85 90
95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Ser Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120
125Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr
Thr Ser 130 135 140Ser Leu Ser Ala Ser
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala145 150
155 160Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Asp 165 170
175Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 195
200 205Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln 210 215 220Gln Gly Asn
Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu225
230 235 240Ile Thr85504PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
85Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1
5 10 15His Ala Ala Arg Pro Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25
30Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr 35 40 45Thr Phe Thr
Asn Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln 50
55 60Gly Leu Glu Trp Met Gly Phe Ile Thr Pro Thr Thr
Gly Tyr Pro Glu65 70 75
80Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser
85 90 95Thr Ser Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100
105 110Ala Val Tyr Tyr Cys Ala Arg Arg Lys Val Gly Lys
Gly Val Tyr Tyr 115 120 125Ala Leu
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130
135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Asp Ile145 150 155
160Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
165 170 175Val Thr Ile Thr
Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 180
185 190Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys
Leu Leu Ile Tyr Asn 195 200 205Thr
Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 210
215 220Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp225 230 235
240Val Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Ser Pro Trp Thr
Phe 245 250 255Gly Gly Gly
Thr Lys Val Glu Ile Lys Glu Gln Lys Leu Ile Ser Glu 260
265 270Glu Asp Leu Asn Gly Ala Ala Thr Thr Thr
Pro Ala Pro Arg Pro Pro 275 280
285Thr Pro Ala Pro Thr Ile Ala Leu Gln Pro Leu Ser Leu Arg Pro Glu 290
295 300Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr Arg Gly Leu Asp305 310
315 320Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly
Gly Val Leu Ala 325 330
335Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
340 345 350Ser Lys Arg Ser Arg Leu
Leu His Ser Asp Tyr Met Asn Met Thr Pro 355 360
365Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
Pro Pro 370 375 380Arg Asp Phe Ala Ala
Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala385 390
395 400Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu 405 410
415Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
420 425 430Arg Asp Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 435
440 445Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser 450 455 460Glu Ile Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly465
470 475 480Leu Tyr Gln Gly Leu Ser Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu 485
490 495His Met Gln Ala Leu Pro Pro Arg
50086817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10
15Gly Ser Thr Gly Ala Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys
20 25 30Gly Gly Ser Glu Val Lys Leu
Gln Glu Ser Gly Pro Gly Leu Val Ala 35 40
45Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser
Leu 50 55 60Pro Asp Tyr Gly Val Ser
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu65 70
75 80Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr
Thr Tyr Tyr Asn Ser 85 90
95Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
100 105 110Val Phe Leu Lys Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 115 120
125Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
Asp Tyr 130 135 140Trp Gly Gln Gly Thr
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser145 150
155 160Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Gln Met Thr Gln 165 170
175Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
180 185 190Cys Arg Ala Ser Gln
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln 195
200 205Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His
Thr Ser Arg Leu 210 215 220His Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp225
230 235 240Tyr Ser Leu Thr Ile Ser Asn
Leu Glu Gln Glu Asp Ile Ala Thr Tyr 245
250 255Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gly Gly Thr 260 265 270Lys
Leu Glu Ile Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275
280 285Ala Pro Thr Ile Ala Leu Gln Pro Leu
Ser Leu Arg Pro Glu Ala Cys 290 295
300Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala305
310 315 320Cys Asp Gly Pro
Ala Gly Ser Leu Leu Gly Ser Gly Gln Met Gln Ile 325
330 335Thr Leu Trp Gly Ser Leu Ala Ala Val Ala
Ile Phe Phe Val Ile Thr 340 345
350Phe Leu Ile Phe Leu Cys Ser Ser Cys Asp Arg Glu Lys Lys Pro Arg
355 360 365Ser Ala Ile Gln Ala Ala Trp
Pro Ser Gly Thr Glu Cys Ile Ala Lys 370 375
380Tyr Asn Phe His Gly Thr Ala Glu Gln Asp Leu Pro Phe Cys Lys
Gly385 390 395 400Asp Val
Leu Thr Ile Val Ala Val Thr Lys Asp Pro Asn Trp Tyr Lys
405 410 415Ala Lys Asn Lys Val Gly Arg
Glu Gly Ile Ile Pro Ala Asn Tyr Val 420 425
430Gln Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser Leu
Met Pro 435 440 445Trp Phe His Gly
Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu Leu Tyr 450
455 460Pro Pro Glu Thr Gly Leu Phe Leu Val Arg Glu Ser
Thr Asn Tyr Pro465 470 475
480Gly Asp Tyr Thr Leu Cys Val Ser Cys Asp Gly Lys Val Glu His Tyr
485 490 495Arg Ile Met Tyr His
Ala Ser Lys Leu Ser Ile Asp Glu Glu Val Tyr 500
505 510Phe Glu Asn Leu Met Gln Leu Val Glu His Tyr Thr
Ser Asp Ala Asp 515 520 525Gly Leu
Cys Thr Arg Leu Ile Lys Pro Lys Val Met Glu Gly Thr Val 530
535 540Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp
Ala Leu Asn Met Lys545 550 555
560Glu Leu Lys Leu Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly Asp Val
565 570 575Met Leu Gly Asp
Tyr Arg Gly Asn Lys Val Ala Val Lys Cys Ile Lys 580
585 590Asn Asp Ala Thr Ala Gln Ala Phe Leu Ala Glu
Ala Ser Val Met Thr 595 600 605Gln
Leu Arg His Ser Asn Leu Val Gln Leu Leu Gly Val Ile Val Glu 610
615 620Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu
Tyr Met Ala Lys Gly Ser625 630 635
640Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu Gly Gly
Asp 645 650 655Cys Leu Leu
Lys Phe Ser Leu Asp Val Cys Glu Ala Met Glu Tyr Leu 660
665 670Glu Gly Asn Asn Phe Val His Arg Asp Leu
Ala Ala Arg Asn Val Leu 675 680
685Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr Lys 690
695 700Glu Ala Ser Ser Thr Gln Asp Thr
Gly Lys Leu Pro Val Lys Trp Thr705 710
715 720Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr
Lys Ser Asp Val 725 730
735Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Tyr Ser Phe Gly Arg Val
740 745 750Pro Tyr Pro Arg Ile Pro
Leu Lys Asp Val Val Pro Arg Val Glu Lys 755 760
765Gly Tyr Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val
Tyr Glu 770 775 780Val Met Lys Asn Cys
Trp His Leu Asp Ala Ala Met Arg Pro Ser Phe785 790
795 800Leu Gln Leu Arg Glu Gln Leu Glu His Ile
Lys Thr His Glu Leu His 805 810
815Leu871015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10
15Gly Ser Thr Gly Ala Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys
20 25 30Gly Gly Ser Glu Val Lys Leu
Gln Glu Ser Gly Pro Gly Leu Val Ala 35 40
45Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser
Leu 50 55 60Pro Asp Tyr Gly Val Ser
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu65 70
75 80Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr
Thr Tyr Tyr Asn Ser 85 90
95Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
100 105 110Val Phe Leu Lys Met Asn
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 115 120
125Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
Asp Tyr 130 135 140Trp Gly Gln Gly Thr
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser145 150
155 160Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Gln Met Thr Gln 165 170
175Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
180 185 190Cys Arg Ala Ser Gln
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln 195
200 205Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His
Thr Ser Arg Leu 210 215 220His Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp225
230 235 240Tyr Ser Leu Thr Ile Ser Asn
Leu Glu Gln Glu Asp Ile Ala Thr Tyr 245
250 255Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gly Gly Thr 260 265 270Lys
Leu Glu Ile Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275
280 285Ala Pro Thr Ile Ala Leu Gln Pro Leu
Ser Leu Arg Pro Glu Ala Cys 290 295
300Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala305
310 315 320Cys Asp Phe Trp
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 325
330 335Ser Leu Leu Val Thr Val Ala Phe Ile Ile
Phe Trp Val Ser Ala Ile 340 345
350Gln Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile Ala Lys Tyr Asn Phe
355 360 365His Gly Thr Ala Glu Gln Asp
Leu Pro Phe Cys Lys Gly Asp Val Leu 370 375
380Thr Ile Val Ala Val Thr Lys Asp Pro Asn Trp Tyr Lys Ala Lys
Asn385 390 395 400Lys Val
Gly Arg Glu Gly Ile Ile Pro Ala Asn Tyr Val Gln Lys Arg
405 410 415Glu Gly Val Lys Ala Gly Thr
Lys Leu Ser Leu Met Pro Trp Phe His 420 425
430Gly Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu Leu Tyr Pro
Pro Glu 435 440 445Thr Gly Leu Phe
Leu Val Arg Glu Ser Thr Asn Tyr Pro Gly Asp Tyr 450
455 460Thr Leu Cys Val Ser Cys Asp Gly Lys Val Glu His
Tyr Arg Ile Met465 470 475
480Tyr His Ala Ser Lys Leu Ser Ile Asp Glu Glu Val Tyr Phe Glu Asn
485 490 495Leu Met Gln Leu Val
Glu His Tyr Thr Ser Asp Ala Asp Gly Leu Cys 500
505 510Thr Arg Leu Ile Lys Pro Lys Val Met Glu Gly Thr
Val Ala Ala Gln 515 520 525Asp Glu
Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys Glu Leu Lys 530
535 540Leu Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly
Asp Val Met Leu Gly545 550 555
560Asp Tyr Arg Gly Asn Lys Val Ala Val Lys Cys Ile Lys Asn Asp Ala
565 570 575Thr Ala Gln Ala
Phe Leu Ala Glu Ala Ser Val Met Thr Gln Leu Arg 580
585 590His Ser Asn Leu Val Gln Leu Leu Gly Val Ile
Val Glu Glu Lys Gly 595 600 605Gly
Leu Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly Ser Leu Val Asp 610
615 620Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu
Gly Gly Asp Cys Leu Leu625 630 635
640Lys Phe Ser Leu Asp Val Cys Glu Ala Met Glu Tyr Leu Glu Gly
Asn 645 650 655Asn Phe Val
His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Ser Glu 660
665 670Asp Asn Val Ala Lys Val Ser Asp Phe Gly
Leu Thr Lys Glu Ala Ser 675 680
685Ser Thr Gln Asp Thr Gly Lys Leu Pro Val Lys Trp Thr Ala Pro Glu 690
695 700Ala Leu Arg Glu Lys Lys Phe Ser
Thr Lys Ser Asp Val Trp Ser Phe705 710
715 720Gly Ile Leu Leu Trp Glu Ile Tyr Ser Phe Gly Arg
Val Pro Tyr Pro 725 730
735Arg Ile Pro Leu Lys Asp Val Val Pro Arg Val Glu Lys Gly Tyr Lys
740 745 750Met Asp Ala Pro Asp Gly
Cys Pro Pro Ala Val Tyr Glu Val Met Lys 755 760
765Asn Cys Trp His Leu Asp Ala Ala Met Arg Pro Ser Phe Leu
Gln Leu 770 775 780Arg Glu Gln Leu Glu
His Ile Lys Thr His Glu Leu His Leu Glu Gly785 790
795 800Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro 805 810
815Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val
820 825 830Pro Arg Ala Val Arg
Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala 835
840 845Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu
Arg Val Thr Glu 850 855 860Leu Gln Glu
Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val865
870 875 880Trp Val Ala Asp Asp Gly Ala
Ala Val Ala Val Trp Thr Thr Pro Glu 885
890 895Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly
Pro Arg Met Ala 900 905 910Glu
Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu 915
920 925Leu Ala Pro His Arg Pro Lys Glu Pro
Ala Trp Phe Leu Ala Thr Val 930 935
940Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val945
950 955 960Leu Pro Gly Val
Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu 965
970 975Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe
Tyr Glu Arg Leu Gly Phe 980 985
990Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys
995 1000 1005Met Thr Arg Lys Pro Gly
Ala 1010 101588946PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 88Met Glu Thr Asp Thr Leu
Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5
10 15Gly Ser Thr Gly Ala Gly Gly Ser Asp Tyr Lys Asp
Asp Asp Asp Lys 20 25 30Gly
Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 35
40 45Pro Ser Gln Ser Leu Ser Val Thr Cys
Thr Val Ser Gly Val Ser Leu 50 55
60Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu65
70 75 80Glu Trp Leu Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 85
90 95Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp
Asn Ser Lys Ser Gln 100 105
110Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
115 120 125Tyr Cys Ala Lys His Tyr Tyr
Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 130 135
140Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
Ser145 150 155 160Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
165 170 175Thr Thr Ser Ser Leu Ser Ala
Ser Leu Gly Asp Arg Val Thr Ile Ser 180 185
190Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr
Gln Gln 195 200 205Lys Pro Asp Gly
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu 210
215 220His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp225 230 235
240Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr
245 250 255Phe Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr 260
265 270Lys Leu Glu Ile Thr Thr Thr Thr Pro Ala Pro Arg
Pro Pro Thr Pro 275 280 285Ala Pro
Thr Ile Ala Leu Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 290
295 300Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala305 310 315
320Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
325 330 335Ser Leu Leu Val
Thr Val Ala Phe Ile Ile Phe Trp Val Val Lys Ala 340
345 350Gly Thr Lys Leu Ser Leu Met Pro Trp Phe His
Gly Lys Ile Thr Arg 355 360 365Glu
Gln Ala Glu Arg Leu Leu Tyr Pro Pro Glu Thr Gly Leu Phe Leu 370
375 380Val Arg Glu Ser Thr Asn Tyr Pro Gly Asp
Tyr Thr Leu Cys Val Ser385 390 395
400Cys Asp Gly Lys Val Glu His Tyr Arg Ile Met Tyr His Ala Ser
Lys 405 410 415Leu Ser Ile
Asp Glu Glu Val Tyr Phe Glu Asn Leu Met Gln Leu Val 420
425 430Glu His Tyr Thr Ser Asp Ala Asp Gly Leu
Cys Thr Arg Leu Ile Lys 435 440
445Pro Lys Val Met Glu Gly Thr Val Ala Ala Gln Asp Glu Phe Tyr Arg 450
455 460Ser Gly Trp Ala Leu Asn Met Lys
Glu Leu Lys Leu Leu Gln Thr Ile465 470
475 480Gly Lys Gly Glu Phe Gly Asp Val Met Leu Gly Asp
Tyr Arg Gly Asn 485 490
495Lys Val Ala Val Lys Cys Ile Lys Asn Asp Ala Thr Ala Gln Ala Phe
500 505 510Leu Ala Glu Ala Ser Val
Met Thr Gln Leu Arg His Ser Asn Leu Val 515 520
525Gln Leu Leu Gly Val Ile Val Glu Glu Lys Gly Gly Leu Tyr
Ile Val 530 535 540Thr Glu Tyr Met Ala
Lys Gly Ser Leu Val Asp Tyr Leu Arg Ser Arg545 550
555 560Gly Arg Ser Val Leu Gly Gly Asp Cys Leu
Leu Lys Phe Ser Leu Asp 565 570
575Val Cys Glu Ala Met Glu Tyr Leu Glu Gly Asn Asn Phe Val His Arg
580 585 590Asp Leu Ala Ala Arg
Asn Val Leu Val Ser Glu Asp Asn Val Ala Lys 595
600 605Val Ser Asp Phe Gly Leu Thr Lys Glu Ala Ser Ser
Thr Gln Asp Thr 610 615 620Gly Lys Leu
Pro Val Lys Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys625
630 635 640Lys Phe Ser Thr Lys Ser Asp
Val Trp Ser Phe Gly Ile Leu Leu Trp 645
650 655Glu Ile Tyr Ser Phe Gly Arg Val Pro Tyr Pro Arg
Ile Pro Leu Lys 660 665 670Asp
Val Val Pro Arg Val Glu Lys Gly Tyr Lys Met Asp Ala Pro Asp 675
680 685Gly Cys Pro Pro Ala Val Tyr Glu Val
Met Lys Asn Cys Trp His Leu 690 695
700Asp Ala Ala Met Arg Pro Ser Phe Leu Gln Leu Arg Glu Gln Leu Glu705
710 715 720His Ile Lys Thr
His Glu Leu His Leu Glu Gly Arg Gly Ser Leu Leu 725
730 735Thr Cys Gly Asp Val Glu Glu Asn Pro Gly
Pro Met Thr Glu Tyr Lys 740 745
750Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val Pro Arg Ala Val Arg
755 760 765Thr Leu Ala Ala Ala Phe Ala
Asp Tyr Pro Ala Thr Arg His Thr Val 770 775
780Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu Leu Gln Glu Leu
Phe785 790 795 800Leu Thr
Arg Val Gly Leu Asp Ile Gly Lys Val Trp Val Ala Asp Asp
805 810 815Gly Ala Ala Val Ala Val Trp
Thr Thr Pro Glu Ser Val Glu Ala Gly 820 825
830Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala Glu Leu Ser
Gly Ser 835 840 845Arg Leu Ala Ala
Gln Gln Gln Met Glu Gly Leu Leu Ala Pro His Arg 850
855 860Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val Gly
Val Ser Pro Asp865 870 875
880His Gln Gly Lys Gly Leu Gly Ser Ala Val Val Leu Pro Gly Val Glu
885 890 895Ala Ala Glu Arg Ala
Gly Val Pro Ala Phe Leu Glu Thr Ser Ala Pro 900
905 910Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe Thr
Val Thr Ala Asp 915 920 925Val Glu
Val Pro Glu Gly Pro Arg Thr Trp Cys Met Thr Arg Lys Pro 930
935 940Gly Ala945891020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Ala Gly
Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 20 25
30Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala 35 40 45Pro Ser Gln
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 50
55 60Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro
Arg Lys Gly Leu65 70 75
80Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
85 90 95Ala Leu Lys Ser Arg Leu
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 100
105 110Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Ile Tyr 115 120 125Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 130
135 140Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser145 150 155
160Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
165 170 175Thr Thr Ser Ser
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 180
185 190Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu
Asn Trp Tyr Gln Gln 195 200 205Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu 210
215 220His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp225 230 235
240Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr 245 250 255Phe Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr 260
265 270Lys Leu Glu Ile Thr Thr Thr Thr Pro Ala
Pro Arg Pro Pro Thr Pro 275 280
285Ala Pro Thr Ile Ala Leu Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 290
295 300Arg Pro Ala Ala Gly Gly Ala Val
His Thr Arg Gly Leu Asp Phe Ala305 310
315 320Cys Asp Glu Glu Ala Ile Leu Val Pro Cys Val Leu
Gly Leu Leu Leu 325 330
335Leu Pro Ile Leu Ala Met Leu Met Ala Leu Cys Val His Cys His Arg
340 345 350Leu Pro Ser Ala Ile Gln
Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile 355 360
365Ala Lys Tyr Asn Phe His Gly Thr Ala Glu Gln Asp Leu Pro
Phe Cys 370 375 380Lys Gly Asp Val Leu
Thr Ile Val Ala Val Thr Lys Asp Pro Asn Trp385 390
395 400Tyr Lys Ala Lys Asn Lys Val Gly Arg Glu
Gly Ile Ile Pro Ala Asn 405 410
415Tyr Val Gln Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser Leu
420 425 430Met Pro Trp Phe His
Gly Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu 435
440 445Leu Tyr Pro Pro Glu Thr Gly Leu Phe Leu Val Arg
Glu Ser Thr Asn 450 455 460Tyr Pro Gly
Asp Tyr Thr Leu Cys Val Ser Cys Asp Gly Lys Val Glu465
470 475 480His Tyr Arg Ile Met Tyr His
Ala Ser Lys Leu Ser Ile Asp Glu Glu 485
490 495Val Tyr Phe Glu Asn Leu Met Gln Leu Val Glu His
Tyr Thr Ser Asp 500 505 510Ala
Asp Gly Leu Cys Thr Arg Leu Ile Lys Pro Lys Val Met Glu Gly 515
520 525Thr Val Ala Ala Gln Asp Glu Phe Tyr
Arg Ser Gly Trp Ala Leu Asn 530 535
540Met Lys Glu Leu Lys Leu Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly545
550 555 560Asp Val Met Leu
Gly Asp Tyr Arg Gly Asn Lys Val Ala Val Lys Cys 565
570 575Ile Lys Asn Asp Ala Thr Ala Gln Ala Phe
Leu Ala Glu Ala Ser Val 580 585
590Met Thr Gln Leu Arg His Ser Asn Leu Val Gln Leu Leu Gly Val Ile
595 600 605Val Glu Glu Lys Gly Gly Leu
Tyr Ile Val Thr Glu Tyr Met Ala Lys 610 615
620Gly Ser Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu
Gly625 630 635 640Gly Asp
Cys Leu Leu Lys Phe Ser Leu Asp Val Cys Glu Ala Met Glu
645 650 655Tyr Leu Glu Gly Asn Asn Phe
Val His Arg Asp Leu Ala Ala Arg Asn 660 665
670Val Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe
Gly Leu 675 680 685Thr Lys Glu Ala
Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val Lys 690
695 700Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe
Ser Thr Lys Ser705 710 715
720Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Tyr Ser Phe Gly
725 730 735Arg Val Pro Tyr Pro
Arg Ile Pro Leu Lys Asp Val Val Pro Arg Val 740
745 750Glu Lys Gly Tyr Lys Met Asp Ala Pro Asp Gly Cys
Pro Pro Ala Val 755 760 765Tyr Glu
Val Met Lys Asn Cys Trp His Leu Asp Ala Ala Met Arg Pro 770
775 780Ser Phe Leu Gln Leu Arg Glu Gln Leu Glu His
Ile Lys Thr His Glu785 790 795
800Leu His Leu Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
805 810 815Glu Asn Pro Gly
Pro Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala 820
825 830Thr Arg Asp Asp Val Pro Arg Ala Val Arg Thr
Leu Ala Ala Ala Phe 835 840 845Ala
Asp Tyr Pro Ala Thr Arg His Thr Val Asp Pro Asp Arg His Ile 850
855 860Glu Arg Val Thr Glu Leu Gln Glu Leu Phe
Leu Thr Arg Val Gly Leu865 870 875
880Asp Ile Gly Lys Val Trp Val Ala Asp Asp Gly Ala Ala Val Ala
Val 885 890 895Trp Thr Thr
Pro Glu Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile 900
905 910Gly Pro Arg Met Ala Glu Leu Ser Gly Ser
Arg Leu Ala Ala Gln Gln 915 920
925Gln Met Glu Gly Leu Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp 930
935 940Phe Leu Ala Thr Val Gly Val Ser
Pro Asp His Gln Gly Lys Gly Leu945 950
955 960Gly Ser Ala Val Val Leu Pro Gly Val Glu Ala Ala
Glu Arg Ala Gly 965 970
975Val Pro Ala Phe Leu Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr
980 985 990Glu Arg Leu Gly Phe Thr
Val Thr Ala Asp Val Glu Val Pro Glu Gly 995 1000
1005Pro Arg Thr Trp Cys Met Thr Arg Lys Pro Gly Ala
1010 1015 1020901034PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
90Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Ala Gly
Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 20 25
30Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala 35 40 45Pro Ser Gln
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 50
55 60Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro
Arg Lys Gly Leu65 70 75
80Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
85 90 95Ala Leu Lys Ser Arg Leu
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 100
105 110Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Ile Tyr 115 120 125Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 130
135 140Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser145 150 155
160Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
165 170 175Thr Thr Ser Ser
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 180
185 190Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu
Asn Trp Tyr Gln Gln 195 200 205Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu 210
215 220His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp225 230 235
240Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr 245 250 255Phe Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr 260
265 270Lys Leu Glu Ile Thr Thr Thr Thr Pro Ala
Pro Arg Pro Pro Thr Pro 275 280
285Ala Pro Thr Ile Ala Leu Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 290
295 300Arg Pro Ala Ala Gly Gly Ala Val
His Thr Arg Gly Leu Asp Phe Ala305 310
315 320Cys Asp Gly Pro Ala Gly Ser Leu Leu Gly Ser Gly
Gln Met Gln Ile 325 330
335Thr Leu Trp Gly Ser Leu Ala Ala Val Ala Ile Phe Phe Val Ile Thr
340 345 350Phe Leu Ile Phe Leu Cys
Ser Ser Cys Asp Arg Glu Lys Lys Pro Arg 355 360
365Ser Ala Ile Gln Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile
Ala Lys 370 375 380Tyr Asn Phe His Gly
Thr Ala Glu Gln Asp Leu Pro Phe Cys Lys Gly385 390
395 400Asp Val Leu Thr Ile Val Ala Val Thr Lys
Asp Pro Asn Trp Tyr Lys 405 410
415Ala Lys Asn Lys Val Gly Arg Glu Gly Ile Ile Pro Ala Asn Tyr Val
420 425 430Gln Lys Arg Glu Gly
Val Lys Ala Gly Thr Lys Leu Ser Leu Met Pro 435
440 445Trp Phe His Gly Lys Ile Thr Arg Glu Gln Ala Glu
Arg Leu Leu Tyr 450 455 460Pro Pro Glu
Thr Gly Leu Phe Leu Val Arg Glu Ser Thr Asn Tyr Pro465
470 475 480Gly Asp Tyr Thr Leu Cys Val
Ser Cys Asp Gly Lys Val Glu His Tyr 485
490 495Arg Ile Met Tyr His Ala Ser Lys Leu Ser Ile Asp
Glu Glu Val Tyr 500 505 510Phe
Glu Asn Leu Met Gln Leu Val Glu His Tyr Thr Ser Asp Ala Asp 515
520 525Gly Leu Cys Thr Arg Leu Ile Lys Pro
Lys Val Met Glu Gly Thr Val 530 535
540Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys545
550 555 560Glu Leu Lys Leu
Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly Asp Val 565
570 575Met Leu Gly Asp Tyr Arg Gly Asn Lys Val
Ala Val Lys Cys Ile Lys 580 585
590Asn Asp Ala Thr Ala Gln Ala Phe Leu Ala Glu Ala Ser Val Met Thr
595 600 605Gln Leu Arg His Ser Asn Leu
Val Gln Leu Leu Gly Val Ile Val Glu 610 615
620Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly
Ser625 630 635 640Leu Val
Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu Gly Gly Asp
645 650 655Cys Leu Leu Lys Phe Ser Leu
Asp Val Cys Glu Ala Met Glu Tyr Leu 660 665
670Glu Gly Asn Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn
Val Leu 675 680 685Val Ser Glu Asp
Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr Lys 690
695 700Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro
Val Lys Trp Thr705 710 715
720Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys Ser Asp Val
725 730 735Trp Ser Phe Gly Ile
Leu Leu Trp Glu Ile Tyr Ser Phe Gly Arg Val 740
745 750Pro Tyr Pro Arg Ile Pro Leu Lys Asp Val Val Pro
Arg Val Glu Lys 755 760 765Gly Tyr
Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr Glu 770
775 780Val Met Lys Asn Cys Trp His Leu Asp Ala Ala
Met Arg Pro Ser Phe785 790 795
800Leu Gln Leu Arg Glu Gln Leu Glu His Ile Lys Thr His Glu Leu His
805 810 815Leu Glu Gly Arg
Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn 820
825 830Pro Gly Pro Met Thr Glu Tyr Lys Pro Thr Val
Arg Leu Ala Thr Arg 835 840 845Asp
Asp Val Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp 850
855 860Tyr Pro Ala Thr Arg His Thr Val Asp Pro
Asp Arg His Ile Glu Arg865 870 875
880Val Thr Glu Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp
Ile 885 890 895Gly Lys Val
Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr 900
905 910Thr Pro Glu Ser Val Glu Ala Gly Ala Val
Phe Ala Glu Ile Gly Pro 915 920
925Arg Met Ala Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met 930
935 940Glu Gly Leu Leu Ala Pro His Arg
Pro Lys Glu Pro Ala Trp Phe Leu945 950
955 960Ala Thr Val Gly Val Ser Pro Asp His Gln Gly Lys
Gly Leu Gly Ser 965 970
975Ala Val Val Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro
980 985 990Ala Phe Leu Glu Thr Ser
Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg 995 1000
1005Leu Gly Phe Thr Val Thr Ala Asp Val Glu Val Pro
Glu Gly Pro 1010 1015 1020Arg Thr Trp
Cys Met Thr Arg Lys Pro Gly Ala 1025 1030
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